Morpholino oligonucleotide manufacturing method

ABSTRACT

Using a morpholino nucleotide wherein 5′-hydroxy group or a hydroxy group present on the substituent of the 5′-hydroxy group is protected by a protecting group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms as a starting material, a method capable of efficiently producing the morpholino oligonucleotide in a high yield by a liquid phase synthesis can be provided.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/JP2014/063750, filed on May 23, 2014, and claims priority toJapanese Patent Application No. 2013-110358, filed on May 24, 2013, allof which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a production method of morpholinooligonucleotide, and morpholino nucleotide used as a starting materialof the production method.

Discussion of the Background

Morpholino oligonucleotide is a compound attracting attention for itsuse as an antisense oligonucleotide, since it shows high affinity forDNA and RNA, resistance to various nucleases, stability in vivo and lowtoxicity (see non-patent document 1).

As a production method of morpholino oligonucleotide, a solid phasesynthesis and a liquid phase synthesis have been reported (seenon-patent document 2, patent documents 1-5).

The solid phase synthesis is advantageous from the aspect of speed sinceit enables automatic synthesis. On the contrary, it is not suitable forindustrial large scale synthesis since scaling-up is limited due tofacility restriction, and low reactivity requires use of an excessmonomer to be the reagent in a nucleotide elongation reaction. Also, itis associated with defects in that confirmation of the progress statusof the reaction in an intermediate stage, analysis of intermediatestructure and the like are difficult.

On the other hand, the liquid phase synthesis requires complicatedtreatments such as column purification and the like, and a large-scaleand rapid synthesis of about 20 mer chain length morpholinooligonucleotide, which is utilizable as an antisense pharmaceuticalproduct, has been difficult.

In recent years, a synthetic method using hydrophobic group-linkednucleoside, pseudo solid phase-protected nucleoside and the like hasbeen reported as an attempt to solve the respective defects of theliquid phase method and the solid phase method (see patent documents 6,7). However, synthesis of morpholino oligonucleotide different in thereaction pathway and the reaction itself from those of oligonucleotidesynthesis is not described or suggested.

DOCUMENT LIST Patent Document

-   patent document 1: WO 91/09033-   patent document 2: WO 2006/008113-   patent document 3: US 2009/0131632 A1-   patent document 4: WO 2009/064471-   patent document 5: WO 2012/043730-   patent document 6: JP-A-2010-275254-   patent document 7: WO 2012/157723

Non-Patent Document

-   non-patent document 1: Summerton, J. et al., Antisense and Nucleic    Acid Drug Development, 1997, Vol. 7, p. 187.-   non-patent document 2: Harakawa et al., Bioorganic & Medicinal    Chemistry Letters, 2012, Vol. 22, p. 1445-1447

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The problem of the present invention is to provide a method of producinga morpholino oligonucleotide efficiently and in a high yield by a liquidphase method, and a novel morpholino nucleoside to be a startingmaterial of the method.

Means of Solving the Problems

The present inventors have found that the above-mentioned problem can besolved by protecting the 5′-hydroxy group side with a particularprotecting group in liquid phase synthesis of a morpholinooligonucleotide.

That is, using a morpholino nucleotide having the 5′-hydroxy group sideprotected by the protecting group as a starting material of morpholinooligonucleotide liquid phase synthesis, condensation reaction itself canbe performed in a liquid phase, the reactivity is remarkably improved ascompared to the solid phase method, monomer equivalents to be used canbe remarkably reduced and, after the reaction, morpholinooligonucleotide can be conveniently isolated and purified by acrystallization or extraction operation. Consequently, a morpholinooligonucleotide having an about 20 mer chain length and utilizable forpharmaceutical products can be synthesized efficiently in a high yieldby a liquid phase method.

On the other hand, in the course of study of liquid phase synthesis ofmorpholino oligonucleotide by the present inventors, a problem was foundthat when an activated morpholino nucleoside monomer to be used for acondensation reaction remains in a trace amount in the subsequent cycleof condensation reaction, double addition is induced, and the quality ofthe obtained morpholino oligonucleotide is degraded. In this regard,they have found that the problem can be solved by treating the activatedmorpholino nucleoside monomer remaining after the condensation reactionwith a quenching agent.

That is, the present invention includes the following.

-   [1] A morpholino nucleotide represented by the formula (I):

-   [wherein-   m is any integer of not less than 0,-   Base in the number of m+1 are each independently an optionally    protected nucleic acid base,-   P¹ is a hydrogen atom, or a temporary protecting group removable    under acidic conditions,-   X in the number of m are each independently a C₁₋₆ alkoxy group, a    di-C₁₋₆ alkylamino group, or a piperazino group wherein a nitrogen    atom at the 4-position is protected by a protecting group and    further optionally substituted,-   W in the number of m are each independently an oxygen atom or a    sulfur acorn,-   S¹ is a single bond, or a group represented by *O—S²** (wherein *    indicates the bonding position to L, ** indicates the bonding    position to a 5′-hydroxy group, and S² is a spacer having a main    chain containing 1 to 20 atoms),-   L is a single bond, or a group represented by the formula (a1):

-   [wherein * indicates the bonding position to Y;-   ** indicates the bonding position to S¹;-   L₁ is an optionally substituted divalent C₁₋₂₂ hydrocarbon group;    and-   L₂ is C(═O) or a group represented by ***N(R³)—R¹—N(R²)C(−O)**-   (wherein ** indicates the bonding position to L¹, *** indicates the    bonding position to Y, R¹ is an optionally substituted C₁₋₂₂    alkylene group, R² and R³ are each independently a hydrogen atom or    an optionally substituted C₁₋₂₂ alkyl group, or R² and R³ are    optionally joined to form an optionally substituted C₁₋₂₂ alkylene    bond)],-   Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen    atom, an alkyl group or an aralkyl group), and-   Z is a group represented by the formula (a2):

-   [wherein * indicates the bonding position to Y;-   R⁴ is a hydrogen atom, or when R_(b) is a group represented by the    following formula (a3), R⁴ is optionally a single bond or —O— in    combination with R⁶ to form a fluorenyl group or a xanthenyl group    together with ring B;-   Q in the number of k are each independently a single bond, or —O—,    —S—, —OC(═O)—, —NHC(═O)— or —NH—;-   R⁵ in the number of k are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms;-   k is an integer of 1 to 4;-   ring A optionally further has, in addition to QR⁵ in the number of    k, a substituent selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen    atom(s), and a C₁₋₆ alkoxy group optionally substituted by a halogen    atom(s);-   R_(a) is a hydrogen atom;-   R_(b) is a hydrogen atom, or a group represented by the formula    (a3):

-   (wherein * indicates a bonding position;-   j is an integer of 0 to 4;-   Q in the number of j are as defined above;-   R⁷ in the number of j are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms;-   R⁶ is a hydrogen atom, or optionally a single bond or —O— in    combination with R⁴ to form a fluorenyl group or a xanthenyl group    together with ring A; and-   ring B optionally further has, in addition to QR⁷ in the number of    j, a substituent selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen    atom(s), and a C₁₋₆ alkoxy group optionally substituted by a halogen    atom(s)), or-   R_(a) and R_(b) are joined to form an oxygen atom]].-   [2] The morpholino nucleotide of [1], wherein m is 0.-   [3] The morpholino nucleotide of [1] or [2], wherein L in the    formula (I) is a succinyl group, and-   R⁵ and/or R⁷ are/is an alkyl group having 10-40 carbon atoms.-   [4] The morpholino nucleotide of [1] or [2], wherein L in the    formula (I) is a succinyl group, and-   R_(a) and R_(b) are both hydrogen atoms, and R⁵ is an alkyl group    having 10-40 carbon atoms.-   [5] The morpholino nucleotide of [1] or [2], wherein L in the    formula (I) is a succinyl group, and-   R⁵ and/or R⁷ are/is an alkyl group having 12-30 carbon atoms.-   [6] The morpholino nucleotide of [1] or [2], wherein, in the formula    (I), L is a succinyl group, and-   Z—Y— is a group selected from the group consisting of a    3,4,5-tri(octadecyloxy)benzyloxy group,-   a 3,5-di(docosyloxy)benzyloxy group,-   a 3,5-bis[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzyloxy group,-   a 3,4,5-tris[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzyloxy group,-   a 3,4,5-tri(octadecyloxy)benzylamino group,-   a 2,4-di(docosyloxy)benzylamino group,-   a 3,5-di(docosyloxy)benzylamino group,-   a di(4-docosyloxyphenyl)methylamino group,-   a 4-methoxy-2-[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzylamino    group,-   a    4-methoxy-2-[3′,4′,5′-tri(octadecyloxy)cyclohexylmethyloxy]benzylamino    group,-   a 2,4-di(dodecyloxy)benzylamino group,-   a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,-   a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group,-   a 3,5-bis[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzylamino group, and-   a 3,4,5-tris[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzylamino group.-   [7] The morpholino nucleotide of [1] or [2], wherein Z—Y-L- is    selected from the group consisting of-   a 2-{2,4-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a 3,5-di(2′,3′-dihydrophytyloxy)benzylsuccinyl group;-   a 4-(2′,3′-dihydrophytyloxy)benzylsuccinyl group;-   a    2-[1-[(2-chloro-5-(2]′,3′-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl    group;-   a 3,4,5-tri(2′,3′-dihydrophytyloxy)benzylsuccinyl group;-   a 2-{3,4,5-tri(240    ,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;-   a 2-{4-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{2-[3′,4′,5′-tri(2″,3″-dihydrophytyloxy)benzyloxy]-4-methoxybenzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{4-(2′,3′-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl    group;-   a 4-(2′,3′-dihydrophytyloxy)-2-methylbenzylsuccinyl group;-   a    2-{4-(2′,3′-dihydrophytyloxy)-2-methylbenzylaminocarbonyl}ethylcarbonyl    group;-   a 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl    group;-   a    2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl    group;-   a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group;-   a 2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a 2-{3,5-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{1-[2,3,4-tri(2′,3′-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{1-[4-(2′,3′-dihydrophytyloxy)phenyl]-4′-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a 3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylsuccinyl    group; and-   a    2-{3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl    group.-   [8] The morpholino nucleoside of any one of [1]-[7], wherein P¹ is a    trityl group, a monomethoxytrityl group, or a dimethoxytrityl group.-   [9] A method of producing n+p-mer morpholino oligonucleotide,    comprising (1) a step of condensing a p-mer morpholino    oligonucleotide (p is any integer of one or more) wherein a    5′-hydroxy group is activated (thio)phosphated or activated    (thio)phosphoramidated, and a morpholine ring nitrogen atom is    protected by a temporary protecting group removable under acidic    conditions, with an n-mer morpholino oligonucleotide (n is an    integer of one or more) wherein a 5′-hydroxy group or, when the    5′-hydroxy group has a substituent having a hydroxy group, the    hydroxy group present on the substituent is protected by a    protecting group having an alkyl group having not less than 10 and    not more than 300 carbon atoms and/or an alkenyl group having not    less than 10 and not more than 300 carbon atoms, and the morpholine    ring nitrogen atom is not protected, by a (thio)phosphoramidate bond    or (thio)phosphorodiamidate bond via the morpholine ring nitrogen    atom.-   [10] The production method of [9], wherein p is 1.-   [11] The production method of [9] or [10], comprising treating the    reaction mixture with a quenching agent after completion of the    reaction.-   [12] The production method of any one of [9]-[11], further    comprising the following step (1′):-   (1′) a step of removing the temporary protecting group of the    morpholine ring nitrogen atom by reacting, before the condensation    step (1), the n-mer morpholino oligonucleotide wherein the    5′-hydroxy group or, when the 5′-hydroxy group has a substituent    having a hydroxy group, the hydroxy group present on the substituent    is protected by a protecting group having an alkyl group having not    less than 10 and not more than 300 carbon atoms and/or an alkenyl    group having not less than 10 and not more than 300 carbon atoms,    and the morpholine ring nitrogen atom is protected by a temporary    protecting group removable under acidic conditions, with an acid in    a non-polar solvent.-   [13] The production method of [12], wherein the temporary protecting    group is removed in the presence of a cation scavenger.-   [14] The production method of [12] or [13], further comprising a    step of neutralizing with an organic base in step (1′), after    removing the temporary protecting group of the morpholine ring    nitrogen atom.

[15] The production method of any one of [9]-[14], wherein theprotecting group having an alkyl group having not less than 10 and notmore than 300 carbon atoms and/or an alkenyl group having not less than10 and not more than 300 carbon atoms is a group represented by thefollowing formula (II):

Z—Y-L-   (II)

[L is a single bond, or a group represented by the formula (a1):

-   [wherein * indicates the bonding position to Y;-   ** indicates the bonding position to S¹;-   L₁ is an optionally substituted divalent C₁₋₂₂ hydrocarbon group;    and-   L₂ is C(═O) or a group represented by ***N(R³)—R¹—N(R²)C(═O)**-   (wherein ** indicates the bonding position to L¹, *** indicates the    bonding position to Y, R¹ is an optionally substituted C₁₋₂₁    alkylene group. R² and R³ are each independently a hydrogen atom or    an optionally substituted C₁₋₂₀ alkyl group, or R² and R³ are    optionally joined to form an optionally substituted C¹⁻²² alkylene    bond)],-   Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen    atom, an alkyl group or an aralkyl group), and-   Z is a group represented by the formula (a2):

-   [wherein * indicates the bonding position to Y;-   R⁴ is a hydrogen atom, or when R_(b) is a group represented by the    following formula (a3), R⁴ is optionally a single bond or —O— in    combination with R⁶ to form a fluorenyl group or a xanthenyl group    together with ring B;-   Q in the number of k are each independently a single bond, or —O—,    —S—, —OC(═O)—, —NHC(═O)— or —NH—;-   R⁵ in the number of k are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms;-   k is an integer of 1 to 4;-   ring A optionally further has, in addition to QR⁵ in the number of    k, a substituent selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen    atom(s), and a C₁₋₆ alkoxy group optionally substituted by a halogen    atom(s);-   R_(a) is a hydrogen atom;-   R_(b) is a hydrogen atom, or a group represented by the formula    (a3):

-   (wherein * indicates a bonding position;-   j is an integer of 0 to 4;-   Q in the number of j are as defined above;-   R⁷ in the number of j are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms;-   R⁶ is a hydrogen atom, or optionally a single bond or —O— in    combination with R⁴ to form a fluorenyl group or a xanthenyl group    together with ring A; and-   ring B optionally further has, in addition to QR⁷ in the number of    j, a substituent selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen    atom(s), and a C₁₋₆ alkoxy group optionally substituted by a halogen    atom(s)), or-   R_(a) and R_(b) are joined to form an oxygen atom].-   [16] The production method of [15], further comprising the following    step (2):-   (2) a step of obtaining morpholino oligonucleotide by adding a polar    solvent to the reaction mixture obtained in step (1′) and/or (1) and    collecting precipitates thereof by solid-liquid separation.-   [17] The production method of any one of [12]-[16], wherein the    non-polar solvent is selected from the group consisting of a    halogenated solvent, an aromatic solvent, an ester solvent, an    aliphatic solvent, a non-polar ether solvent, and a combination of    these.-   [18] The production method of [16] or [17], wherein the polar    solvent is an alcohol solvent or a nitrile solvent.-   [19] The production method of any one of [9]-[18], further    comprising the following step (3):-   (3) a step of removing all the protecting groups of the obtained    n+p-mer morpholino oligonucleotide.-   [20] The production method of any one of [9]-[19], wherein the    temporary protecting group removable under acidic conditions is a    trityl group, a dimethoxytrityl group, or a monomethoxytrityl group.

Effect of the Invention

According to the present invention, since an advantage of a liquid phasemethod that monomer equivalent of a starting material of a condensationreaction can be reduced by improving the reactivity by protecting the5′-hydroxy group side of morpholino nucleotide with a particularprotecting group, and an advantage of a solid phase method thatmorpholino oligonucleotide can be conveniently isolated and purified bya crystallization, washing or extraction operation in each step of anelongation reaction can be combined, an industrial large-scaleproduction of a morpholino oligonucleotide having about 20 mer chainlength and useful as a pharmaceutical product can be efficientlyperformed in a high yield.

In addition, it was found that degradation of the quality of the objectmorpholino oligonucleotide can be prevented by treating activatedmorpholino nucleoside monomer remaining after condensation reaction witha quenching agent. By combining same with the above-mentioned method, anefficient production method of high quality morpholino oligonucleotidein a large scale can be provided.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a novel morpholino nucleotide whereinthe 5′-hydroxy group side is protected by a particular protecting group,and a morpholine ring nitrogen atom is optionally protected by atemporary protecting group removable under acidic conditions.

Another embodiment of the present invention is a method of producingn+p-mer morpholino oligonucleotide, comprising a step of condensing ap-mer morpholino oligonucleotide (p is any integer of one or more)wherein a 5′-hydroxy group is activated (thio)phosphated or activated(thio)phosphoramidated, and a morpholine ring nitrogen atom is protectedby a temporary protecting group removable under acidic conditions, withan n-mer morpholino oligonucleotide (n is an integer of one or more)wherein a 5′-hydroxy group or a hydroxy group present on a substituentof the 5′-hydroxy group is protected by a protecting group having analkyl group having not less than 10 and not more than 300 carbon atomsand/or an alkenyl group having not less than 10 and not more than 300carbon atoms, and the morpholine ring nitrogen atom is not protected, bya (thio)phosphoramidate bond or (thio)phosphorodiamidate bond via themorpholine ring nitrogen atom.

Explanations are given below.

[Explanation of Terms]

Unless otherwise specified in the sentences, any technical terms andscientific terms used in the present specification, have the samemeaning as those generally understood by those of ordinary skill in theart the present invention belongs to. Any methods and materials similaror equivalent to those described in the present specification can beused for practicing or testing the present invention, and preferablemethods and materials are described in the following. All publicationsand patents referred to in the specification are hereby incorporated byreference so as to describe and disclose constructed products andmethodology described in, for example, publications usable in relationto the described invention.

In the present specification, the “morpholino nucleoside” to be aconstitutional unit of morpholino oligonucleotide is a compoundrepresented by the following formula (1).

(wherein Base is an optionally protected nucleic acid base).

Morpholino nucleoside (1) can be prepared by a method known per se(e.g., the method described in WO 91/09033A1), or a method analogousthereto. Specifically, as shown in the following scheme, thecorresponding ribonucleoside (2) is subjected to oxidative ring openingwith sodium periodate etc. to give the corresponding 2′,3′-dialdehyde(3), the dialdehyde (3) is subjected to ring closure with ammonia togive 2′,3′-dihydroxymorpholino nucleoside (4), and dihydroxymorpholinonucleoside (4) is reduced with a reducing agent (e.g., sodiumcyanoborohydride, sodium triacetoxyborohydride and the like), wherebymorpholino nucleoside (1) can be obtained.

In the present specification, the position numbers (1′,2′ and the like)of morpholino nucleoside correspond to the position numbers of carbonatoms of the ribose of the starting material ribonucleoside (2).

In the present specification/morpholino oligonucleotide means a compoundwherein two or more morpholino nucleosides are polymerized by(thio)phosphoramidate bonding or (thio)phosphorodiamidate bonding via a5′-hydroxy group and the nitrogen atom of a morpholine ring. Forexample, as m′+1-mer morpholino oligonucleotide, a compound representedby the following formula (5) can be mentioned.

-   (wherein-   m′ is any integer of one or more,-   Base in the number of m′+1 are each independently an optionally    protected nucleic acid base,-   X in the number of m′ are each independently a C₁₋₆ alkoxy group, a    di-C₁₋₆ alkylamino group, or a piperazino group wherein a nitrogen    atom at the 4-position is protected by a protecting group, and    further optionally substituted, and the like, and-   W in the number of m′ are each independently an oxygen atom or a    sulfur atom).

In the present specification, the “piperazino group wherein a 4-positionnitrogen atom is protected by a protecting group, and further optionallysubstituted” means that the 4-position nitrogen atom of the piperazinogroup is protected by a protecting group, and a piperazino groupprotected by a protecting group sustainable under the deprotectionconditions of the morpholine ring nitrogen atom of morpholino nucleotideis preferable. As the “protecting group of the 4-position nitrogen atomof the piperazino group”, an acyl group is preferable and, for example,an acyl group having a fluoro group in the carbon chain such asmonofluoroacetyl group, difluoroacetyl group, trifluoroacetyl group,2-fluoropropionyl group, 2,2-difluoropropionyl group,3,3,3-trifluoropropionyl group, 2,3,3,3-tetrafluoropropionyl group,pentafluoropropionyl group and the like is more preferable (see WO2008/008113). In the piperazino group, a hydrogen atom bonded to thecarbon atom of the piperazino group may be substituted, and examples ofthe substituent include an alkyl group (preferably having 1-3 carbonatoms) such as methyl group and the like, and the like.

In the present specification, morpholino nucleoside at the terminal onthe side having a free hydroxy group at the 5′-position of morpholinooligonucleotide (upper left side of the above-mentioned formula (5)) isreferred to as the “5′-terminus”, and morpholino nucleoside at theterminal on the opposite side (lower right side of the above-mentionedformula (5)) is referred to as the “3′-terminus”, according to the usualpractice in the nucleic acid chemistry.

In the present specification, the “nucleic acid base” is notparticularly limited as long as it can be used for the synthesis ofnucleic acid and includes, for example, a pyrimidine base such ascytosyl group, uracil group, thyminyl group and the like, and a purinebase such as adenyl group, guanyl group and the like. The “optionallyprotected nucleic acid base” means, for example, that an amino group maybe protected in an adenyl group, a guanyl group or a cytosyl group,which is a nucleic acid base having an amino group, and a nucleic acidbase wherein the amino group therein is protected by a protecting groupsustainable under the deprotection conditions of the morpholine ringnitrogen atom of the morpholino nucleotide is preferable. The“amino-protecting group” is not particularly limited, and examplesthereof include the protecting groups described in Greene's PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 4th edition, Wiley-Interscience, 2006 andthe like. Specific examples of the “amino-protecting group” include apivaloyl group, a pivaloyloxymethyl group, a trifluoroacetyl group, aphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a4-tert-butylphenoxyacetyl group, an acetyl group, a benzoyl group, anisobutyryl group, a dimethylformamidinyl group, a9-fluorenylmethyloxycarbonyl group and the like. Among them, aphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an acetyl group,a benzoyl group, an isobutyryl group and a dimethylformamidinyl groupare preferable. In addition, the carbonyl group of the nucleic acid baseis optionally protected, and can be protected, for example, by reactingphenol, 2,5-dichlorophenyl, 3-chlorophenol, 3,5-dichlorophenol,2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol,2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol,4-pivaloyloxybenzyl alcohol, 4-nitrophenethyl alcohol,2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol,2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoylchloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride,4-morpholinecarbonyl chloride, diphenylcarbamoyl chloride and the like.In some cases, the carbonyl-protecting group does not need to beparticularly introduced. Moreover, in addition to the above-mentionedgroups, a modified nucleic acid base (e.g., a 8-bromoadenyl group, a8-bromoguanyl group, a 5-bromocytosyl group, a 5-iodocytosyl group, a5-bromouracil group, a 5-iodouracil group, a 5-fluorouracil group, a5-methylcytosyl group, a 8-oxoguanyl group, a hypoxanthinyl group etc.),which is a nucleic acid base substituted by any 1 to 3 substituents(e.g., a halogen atom, an alkyl group, an aralkyl group, an alkoxygroup, an acyl group, an alkoxyalkyl group, a hydroxy group, an aminogroup, monoalkylamino, dialkylamino, carboxy, cyano, nitro etc.) at anyposition(s), are also encompassed in the “nucleic acid base”.

In the present specification, the “halogen atom” means a fluorine atom,a chlorine atom, a bromine atom or iodine atom.

In the present specification, examples of the “alkyl (group)” include alinear or branched chain alkyl group having one or more carbon atoms.When the carbon number is not particularly limited, it is preferably aC₁₋₁₀ alkyl group, more preferably a C₁₋₆ alkyl group. When the carbonnumber is not particularly limited, for example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and thelike are preferable, and methyl and ethyl are particularly preferable.

In the present specification, the “aralkyl (group)” means a C₇₋₂₀aralkyl group, preferably a C₇₋₁₆ aralkyl group (C₆₋₁₀ aryl-C₁₋₆ alkylgroup). Specific preferable examples include benzyl, 1-phenylethyl,2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl,1-naphthylpropyl and the like, and benzyl is particularly preferable.

In the present specification, examples of the “alkoxy (group)” includean alkoxy group having one or more carbon atoms. When the carbon numberis not particularly limited, it is preferably a C₁₋₁₀ alkoxy group, morepreferably a C₁₋₆ alkoxy group. When the carbon number is notparticularly limited, methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like arepreferable, and methoxy and ethoxy are particularly preferable.

In the present specification, examples of the “acyl (group)” include alinear or branched chain C₁₋₆ alkanoyl group, a C₇₋₁₃ aroyl group andthe like. Specific examples thereof include formyl, acetyl, n-propionyl,isopropionyl, n-butyryl, isobutyryl, pivaloyl, valeryl, hexanoyl,benzoyl, naphthoyl, levulinoyl and the like, each of which is optionallysubstituted.

In the present specification, examples of the “alkenyl (group)” includea linear or branched chain alkenyl group and the like. Examples thereofinclude vinyl, 1-propenyl, allyl, isopropenyl, butenyl, isobutenyl andthe like. Among them, a C₂-C₄ alkenyl group is preferable.

In the present specification, preferable examples of the “alkynyl(group)” include a C₂₋₆ alkynyl group and the like. Examples thereofinclude ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl,3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl,2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like. Among them, aC₂-C₄ alkynyl group is preferable.

In the present specification, the “cycloalkyl (group)” means a cyclicalkyl group, and examples thereof include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Amongthem, a C₃-C₆ cycloalkyl group such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like is preferable, and cyclohexyl isparticularly preferable.

In the present specification, the “aryl (group)” means a monocyclicaromatic or polycyclic (fused) aromatic hydrocarbon group. Specificexamples thereof include a C₆₋₁₄ aryl group such as phenyl, 1-naphthyl,2-naphthyl, biphenylyl, 2-anthryl and the like, and the like. Amongthem, a C₆₋₁₀ aryl group is more preferably and phenyl is particularlypreferable.

In the present specification, examples of the “hydrocarbon group”include an aliphatic hydrocarbon group, an aromatic-aliphatichydrocarbon group, a monocyclic saturated hydrocarbon group, an aromatichydrocarbon group and the like, and specific examples thereof includemonovalent groups such as an alkyl group, an alkenyl group, an alkynylgroup, a cycloalkyl group, an aryl group, an aralkyl group and the likeand a divalent group induced therefrom.

In the present specification, the “organic group having a hydrocarbongroup” means a group having the aforementioned “hydrocarbon group”, andthe moiety other than the “hydrocarbon group” of the “organic grouphaving a hydrocarbon group” can be determined freely. For example, theorganic group optionally has, as a linker, a moiety such as —O—, —S—,—COO—, —OCONH—, —CONH— and the like.

In the present specification, the “substituent” of the “optionallysubstituted” encompasses the aforementioned halogen atom, alkyl group,aralkyl group, alkoxy group, acyl group, alkenyl group, alkynyl group,cycloalkyl group, aryl group, as well as hydroxy group, nitro group,cyano group, guanidyl group, carboxy group, alkoxycarbonyl group (thealkoxy moiety is the same as that in the aforementioned alkoxy group),sulfo group, phospho group, alkylthio group (the alkyl moiety is thesame as that in the aforementioned alkyl group), alkylsulfinyl group(the alkyl moiety is the same as that in the aforementioned alkylgroup), alkylsulfonyl group (the alkyl moiety is the same as that in theaforementioned alkyl group), amino group, monoalkylamino group (thealkyl moiety is the same as that in the aforementioned alkyl group),dialkylamino group (the alkyl moiety is the same as that in theaforementioned alkyl group), oxo group and the like.

[Morpholino Nucleotide Wherein 5′-Hydroxy Group or Hydroxy Group Presenton the Substituent of the 5′-Hydroxy Group is Protected by a ParticularProtecting Group, and Morpholine Ring Nitrogen Atom is OptionallyProtected by a Temporary Projecting Group Removable Under AcidicConditions]

Using the morpholino nucleotide wherein 5′-hydroxy group or hydroxygroup present on the substituent of the 5′-hydroxy group is protected bya particular protecting group used in the present invention, aproduction method of a morpholino oligonucleotide suitable for liquidphase synthesis can be provided.

Since high efficiency and high yield can be achieved in the productionmethod of the object morpholino oligonucleotide, a morpholino nucleotidewherein 5′-hydroxy group or hydroxy group present on the substituent ofthe 5′-hydroxy group is protected by a protecting group having an alkylgroup having not less than 10 and not more than 300 carbon atoms and/oran alkenyl group having not less than 10 and not more than 300 carbonatoms is preferable.

“When the 5′-hydroxy group has a substituent” means that the hydrogenatom of the 5′-hydroxy group is substituted by a substituent having ahydroxy group. The “substituent” of the “substituent having a hydroxygroup” is not particularly limited as long as the main chain isconstituted of 1 to 20 atoms. Here, the “main chain” means a shortestatom chain linking the oxygen atom of the 5′-hydroxy group and theoxygen atom of the hydroxy group on the substituent, and the atom chainis optionally further substituted. The atom constituting the main chainis selected from carbon atom, oxygen atom, nitrogen atom, sulfur atom,phosphorus atom and the like. Specific examples of the “substituenthaving a hydroxy group” include organic groups having a hydrocarbongroup such as alkyl group, aralkyl group, acyl group, alkenyl group,alkynyl group, cycloalkyl group, aryl group, alkoxycarbonyl group andthe like, wherein the hydrogen atom on the hydrocarbon group issubstituted by a hydroxy group and the like. In addition, for example,the substituent of the following 5′-hydroxy group disclosed in WO2008/008113 and the like can be mentioned.

Examples of the “alkyl group having not less than 10 and not more than300 carbon atoms and/or alkenyl group having not less than 10 and notmore than 300 carbon atoms” include monovalent groups and divalentgroups induced therefrom. Among them, alkyl group having 10-40 carbonatoms is preferable, and alkyl group having 10-30 carbon atoms isparticularly preferable. The alkyl group and the alkenyl group of the“alkyl group having not less than 10 and not more than 300 carbon atomsand/or alkenyl group having not less than 10 and not more than 300carbon atoms” include linear or branched chain alkyl group, and linearor branched chain alkenyl group. In the production method of the presentinvention, linear alkyl group and linear alkenyl group are preferable,and linear alkyl group is particularly preferable. Specific preferableexamples of the “alkyl group having not less than 10 and not more than300 carbon atoms and/or alkenyl group having not less than 10 and notmore than 300 carbon atoms” include monovalent aliphatic hydrocarbongroup such as decyl group, dodecyl group, tridecyl group, myristylgroup, cetyl group, stearyl group, oleyl group, linolyl group, arachylgroup, behenyl group, isostearyl group and the like, and divalent groupsinduced therefrom.

As the morpholino oligonucleotide wherein a 5′-hydroxy group or ahydroxy group present on a substituent of the 5′-hydroxy group isprotected by a protecting group having an alkyl group having not lessthan 10 and not more than 300 carbon atoms and/or an alkenyl grouphaving not less than 10 and not more than 300 carbon atoms, and themorpholine ring nitrogen atom is optionally protected by a temporaryprotecting group removable under acidic conditions, a morpholinonucleotide wherein the 5′-hydroxy group or the hydroxy group present onthe substituent of the 5′-hydroxy group is protected by a protectinggroup represented by the formula (II): Z—Y-L- is preferable.Specifically, a novel compound represented by the following formula (I)(hereinafter sometimes to be referred to as the compound of the presentinvention) can be mentioned.

The formula (I):

-   [wherein-   m is any integer of not less than 0,-   Base in the number of m+1 are each independently an optionally    protected nucleic acid base,-   P¹ is a hydrogen atom, or a temporary protecting group removable    under acidic conditions,-   X in the number of m are each independently a C₁₋₆ alkoxy group, a    di-C₁₋₆ alkylamino group, or a piperazino group wherein a nitrogen    atom at the 4-position is protected by a protecting group and    further optionally substituted,-   W in the number of m are each independently an oxygen atom or a    sulfur atom,-   S¹ is a single bond, or a group represented by *O—S²**-   (wherein * indicates the bonding position to L, ** indicates the    bonding position to 5′-hydroxy group, and S² is a spacer having a    main chain containing 1 to 20 atoms), and L is a single bond, or a    group represented by the formula (a1):

-   [wherein * indicates the bonding position to Y;-   ** indicates the bonding position to S¹;-   L₁ is an optionally substituted divalent C₁₋₂₂ hydrocarbon group;    and-   L₂ is C(═O) or a group represented by ***N(R³)—R¹—N(R²)C(═O) **-   (wherein ** indicates the bonding position to L¹, *** indicates the    bonding position to Y, R¹ is an optionally substituted C₁₋₂₂    alkylene group, R² and R³ are each independently a hydrogen atom or    an optionally substituted C₁₋₂₂ alkyl group, or R² and R³ are    optionally joined to form an optionally substituted C₁₋₂₂ alkylene    bond)],-   Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen    atom, an alkyl group or an aralkyl group), and-   Z is a group represented by the formula (a2):

-   [wherein * indicates the bonding position to Y;-   R⁴ is a hydrogen atom, or when R_(b) is a group represented by the    following formula (a3), R⁴ is optionally a single bond or —O— in    combination with R⁶ to form a fluorenyl group or a xanthenyl group    together with ring B;-   Q in the number of k are each independently a single bond, or —O—,    —S—, —OC(═O)—, —NHC(═O)— or —NH—;-   R⁵ in the number of k are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms;-   k is an integer of 1 to 4;-   ring A optionally further has, in addition to QR⁵ in the number of    k, a substituent selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen    atom(s), and a C₁₋₆ alkoxy group optionally substituted by a halogen    atom(s);-   R_(a) is a hydrogen atom;-   R_(b) is a hydrogen atom, or a group represented by the formula    (a3):

-   (wherein * indicates a bonding position;-   j is an integer of 0 to 4;-   Q in the number of j are as defined above;-   R⁷ in the number of j are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms;-   R⁶ is a hydrogen atom, or optionally a single bond or —O— in    combination with R⁴ to form a fluorenyl group or a xanthenyl group    together with ring A; and-   ring B optionally further has, in addition to QR⁷ in the number of    j, a substituent selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen    atom(s), and a C₁₋₆ alkoxy group optionally substituted by a halogen    atom(s)), or-   R_(a) and R_(b) are optionally joined to form an oxygen atom.]]

In the below-mentioned production method of the morpholinooligonucleotide of the present invention, the 5′-hydroxy group isactivated (thio)phosphated or activated (thio)phosphoramidated, and themorpholine ring nitrogen atom is bonded to p-mer morpholinooligonucleotide (p is any integer of one or more) protected by atemporary protecting group removable under acidic conditions, wherebythe compound of the present invention can form m+1+p-mer morpholinooligonucleotide (p is any integer of one or more).

When m is 0, the compound of the present invention is understood to be a“morpholino nucleoside”, which is a starting compound of the 5′-terminalin the synthesis of the morpholino oligonucleotide of the presentinvention. In addition, the compound of the present invention alsoencompasses one wherein the morpholine ring nitrogen atom on the3′-terminus side is unprotected (P¹ is a hydrogen atom) in a broadsense.

In the above-mentioned formula (I), m is any integer of not less than 0,preferably, 0. While the upper limit of m is not particularly limited,it is preferably 49 or less, more preferably 29 or less, furtherpreferably 19 or less.

Base in the number of m+1 in the above-mentioned formula (I) are eachindependently an optionally protected nucleic acid base. The “optionallyprotected nucleic acid base” means, for example, that an amino group maybe protected in an adenyl group, a guanyl group or a cytosyl group,which is a nucleic acid base having an amino group, or imide group isoptionally protected in a thyminyl group, an uracil group having acyclic imide group, and a nucleic acid base wherein the amino grouptherein is protected by a protecting group sustainable under thedeprotection conditions of the morpholine ring nitrogen atom ispreferable. The protecting group of the “amino-protecting group” and the“imido-protecting group” is not particularly limited and, for example,any protecting groups described in Greene's PROTECTIVE GROUPS IN ORGANICSYNTHESIS, 4th ed., JOHN WILLY&SONS (2006) and the like can bementioned. Specific examples of such “amino-protecting group” and“imide-protecting group” include a pivaloyl group, a pivaloyloxymethylgroup, a trifluoroacetyl group, a phenoxyacetyl group, a4-isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, anacetyl group, a benzoyl group, an isobutyryl group, adimethylformamidinyl group, a 9-fluorenylmethyloxycarbonyl group and thelike. Among them, a phenoxyacetyl group, a 4-isopropylphenoxyacetylgroup, an acetyl group, a benzoyl group, an isobutyryl group and adimethylformamidinyl group are preferable. In addition, the carbonylgroup of the nucleic acid base is optionally protected, and can beprotected, for example, by reacting phenol, 2,5-dichlorophenol,3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol,4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol,4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol,4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol,2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol,dimethylcarbamoyl chloride, diethylcarbamoyl chloride,ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride,4-morpholinecarbonyl chloride, diphenylcarbamoyl chloride and the like.In some cases, the carbonyl-protecting group does not particularly needto be introduced.

The temporary protecting group P¹ that can be used as the protectinggroup of the morpholine ring nitrogen atom at the 3′-terminus of thepresent invention is not particularly limited as long as it can bedeprotected under acidic conditions and can be used as ahydroxy-protecting group. Examples thereof include a trityl group,9-(9-phenyl)xanthenyl group, a 9-phenylthioxanthenyl group, di(C₁₋₆alkoxy)trityl groups such as a 1,1-bis(4-methoxyphenyl)-1-phenylmethylgroup, a dimethoxytrityl group and the like, mono(C₁₋₁₈ alkoxy)tritylgroups such as 1-(4-methoxyphenyl)-1,1-diphenylmethyl group,monomethoxytrityl group and the like, and the like can be mentioned.Among these, a trityl group, a monomethoxytrityl group and adimethoxytrityl group are preferable, and a dimethoxytrityl group ismore preferable, in view of easiness of deprotection and easyavailability. Among these, a trityl group, a monomethoxytrityl group anda dimethoxytrityl group are preferable, and a trityl group and adimethoxytrityl group are more preferable, in view of easiness ofdeprotection and easy availability.

In the above-mentioned formula (I), X in the number of m are eachindependently a C₁₋₆ alkoxy group, a di-C₁₋₆ alkylamino group, or apiperazino group wherein a nitrogen atom at the 4-position is protectedby a protecting group and further optionally substituted, preferably adi-C₁₋₆ alkylamino group.

As the C₁₋₆ alkoxy group, a methoxy group or an ethoxy group ispreferable, and a methoxy group is more preferable.

As the di-C₁₋₆ alkylamino group, a dimethylamino group, a diethylaminogroup, an N-ethyl-N-methylamino group and the like are preferable, and adimethylamino group is preferable.

As the protecting group of the 4-position nitrogen atom of thepiperazino group, an acyl group is preferable and, for example, an acylgroup having a fluoro group in the carbon chain, such as amonofluoroacetyl group, a difluoroacetyl group, a trifluoroacetyl group,a 2-fluoropropionyl group, a 2,2-difluoropropionyl group, a3,3,3-trifluoropropionyl group, a 2,3,3,3-tetrafluoropropionyl group, apentafluoropropionyl group and the like, is more preferable. While theprotecting group is generally deprotected after completion of theelongation reaction, the amino group of the piperazino group may befurther modified, after deprotection, by a modifying group according tothe method described in WO 2008/008113. Examples of the modifying groupinclude a halogen atom, an alkyl group, an aralkyl group, an alkoxygroup, an acyl group, an alkenyl group, an alkynyl group, a cycloalkylgroup, an aryl group, a hydroxy group, a nitro group, a cyano group, aguanidyl group, a carboxy group, an alkoxycarbonyl group, a sulfo group,a phospho group, an alkylthio group, an alkylsulfinyl group, analkylsulfonyl group and the like. As the modifying group of thepiperazino group, an optionally substituted acyl group is preferable,and an acyl group (e.g., 6-guanidinohexanoyl group) optionallysubstituted by a guanidyl group is more preferable. In the piperazinogroup, the hydrogen atom bonded to the carbon atom of the piperazinogroup may be substituted, and examples of the substituent include analkyl group (preferably having 1-3 carbon atoms) such as a methyl groupand the like, and the like.

W in the number of m is each independently an oxygen atom or a sulfuratom, preferably an oxygen atom.

S¹ in the above-mentioned formula (I) is a single bond, or a grouprepresented by *O—S²** (wherein * indicates the bonding position to L,** indicates the bonding position to 5′-hydroxy group, and S² is aspacer having a main chain containing 1 to 20 atoms), preferably asingle bond.

The “spacer having a main chain having an atomic number of 1 to 20” forS² is, for example, a divalent group formed by removing a hydroxy groupfrom the aforementioned “substituent having a hydroxy group” when the5′-hydroxy group has a substituent.

A preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1),

-   L₁ is an ethylene group or CH₂—O-1,4-phenylene-O—CH₂; and-   L₂ is C(═O), or a group represented by ***N(R³)—R¹—N(R²)C(═O) **    (wherein ** indicates the bonding position to L₁, *** indicates the    bonding position to Y, R¹ is a C₁₋₆ alkylene group, and R² and R³    are each independently a hydrogen atom or an optionally substituted    C₁₋₆ alkyl group, or R² and R³ are optionally joined to form an    optionally substituted C₁₋₆ alkylene bond).

Another preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1),

-   L₁ is an ethylene group; and-   L₂ is C(═O).

Another preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1),

-   L₁ is an ethylene group; and-   the moiety of N(R³)—R¹—N(R²) for L₂ is a piperazinylene group.

Another preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1).

-   L₁ is an ethylene group; and-   L₂ is a group represented by ***N(R³)—R¹—N(R²)C(═O) ** (wherein **    indicates the bending position to L¹, *** indicates the bonding    position to Y, R¹ is a pentylene group or a hexylene group, and R²    and R² are each independently a hydrogen atom or a methyl group).

A particularly preferable example of the above-mentioned linker L is asingle bond or a succinyl group since it is easily available andeconomical.

Y in the above-mentioned formula (I) is a single bond, an oxygen atom,or NR (wherein R is a hydrogen atom, an alkyl group or an aralkylgroup).

In the present specification, the “alkyl group” for R is a C₁₋₃₀ alkylgroup, preferably a C₁₋₁₀ alkyl group, more preferably a C₁₋₆ alkylgroup. Specific preferable examples thereof include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and the like,and methyl and ethyl are particularly preferable.

In the present specification, the “aralkyl group” for R is a C₇₋₃₀aralkyl group, preferably a C₇₋₂₀ aralkyl group, more preferably a C₇₋₁₆aralkyl group (C₆₋₁₀ aryl-C₁₋₆ alkyl group). Specific preferableexamples thereof include benzyl, 1-phenylethyl, 2-phenylethyl,1-phenylpropyl, α-naphthylmethyl, 1-(α-naphthyl)ethyl,2-(α-naphthyl)ethyl, 1-(α-naphthyl)propyl, β-naphthylmethyl,1-(β-naphthyl)ethyl, 2-(β-naphthyl)ethyl, 1-(β-naphthyl)propyl and thelike, and benzyl is particularly preferable.

R is preferably a hydrogen atom, a C₁₋₆ alkyl group or a C₇₋₁₆ aralkylgroup, more preferably a hydrogen atom, methyl, ethyl or benzyl,particularly preferably a hydrogen atom.

Y is preferably a single bond, an oxygen atom or NH.

A preferable embodiment of Z is a group represented by the formula (a2).

The preferable embodiment for Z in the above-mentioned formula (I), thatis, a group represented by the formula (a2) for Z in the above-mentionedformula (I) is a particular benzyl group (in the formula (a2), bothR_(a) and R_(b) are hydrogen atoms, and R⁴ is a hydrogen atom); aparticular benzoyl group (in the formula (a2) wherein R_(a) and R_(b)are joined to form an oxygen atom, and R⁴ is a hydrogen atom); aparticular diphenylmethyl group (in the formula (a2), R_(a) is ahydrogen atom, R⁴ is a hydrogen atom, k is 1 to 3, and R_(b) is a grouprepresented by the formula (a3) (wherein R⁶ is a hydrogen atom, and j is0 or 1)); a particular fluorenyl group (in the formula (a2), R_(a) is ahydrogen atom, k is 1, R_(b) is a group represented by the formula (a3)(wherein j is 0)), and R⁶ is a single bond together with R⁴ to form afluorene ring together with ring A); a particular xanthenyl group (inthe formula (a2), R_(a) is a hydrogen atom, k is 1, R_(b) is a grouprepresented by the formula (a3) (wherein j is 0), and R⁶ is —O— togetherwith R⁴ to form a xanthine ring together with ring A).

In the QR⁵ group in the number of k in the above-mentioned formula (a2),and the QR⁷ group in the number of j in the formula (a3), Q is a singlebond, or —O—, —S—, —OC(═O)—, —NHC(═O)— or —NH—, preferably —O—. The QR⁵group in the number of k, and QR⁷ group in the number of j may be thesame or different.

In the above-mentioned formula (a2), the “R_(a) and R_(b) are joined toform an oxygen atom” means that R_(a) and R_(b) are joined to form acarbonyl group (C(═O)).

The “organic group having an alkyl group having not less than 10 and notmore than 300 carbon atoms and/or an alkenyl group having not less than10 and not more than 300 carbon atoms” for R⁵ or R⁷ is a monovalentorganic group having an alkyl group having not less than 10 and not morethan 300 carbon atoms and/or an alkenyl group having not less than 10and not more than 300 carbon atoms in the molecule structure thereof.

The carbon number of the “alkyl group having not less than 10 and notmore than 300 carbon atoms and/or alkenyl group having not loss than 10and not more than 300 carbon atoms” of the “organic group having analkyl group having not less than 10 and not more than 300 carbon atomsand/or an alkenyl group having not less than 10 and not more than 300carbon atoms” is preferably 14-40, more preferably 14-30.

The moiety of the “alkyl group having not less than 10 and not more than300 carbon atoms and/or alkenyl group having not less than 10 and notmore than 300 carbon atoms” of the “organic group having an alkyl grouphaving not less than 10 and not more than 300 carbon atoms and/or analkenyl group having not less than 10 and not more than 300 carbonatoms” is not particularly limited, and it may be present at theterminus (monovalent group), or the other site (e.g., divalent group).

As the “alkyl group having not less than 10 and not more than 300 carbonatoms and/or alkenyl group having not less than 10 and not more than 300carbon atoms”, a monovalent group and a divalent group induced therefromcan be mentioned. Among them, an alkyl group having 14-40 carbon atomsis preferable, and an alkyl group having 14-30 carbon atoms isparticularly preferable. Specific examples of the “alkyl group havingnot less than 10 and not more than 300 carbon atoms and/or alkenyl grouphaving not less than 10 and not more than 300 carbon atoms” includemonovalent linear aliphatic hydrocarbon groups such as a decyl group, adodecyl group, a tridecyl group, a myristyl group, a cetyl group, astearyl group, an oleyl group, a linolyl group., an arachyl group, abehenyl group and the like, monovalent branched chain aliphatichydrocarbon groups such as a 3,7,11,15-tetramethylhexadecyl group, a3,7,11-trimethyldodecyl group, a 2,2,4,8,10,10-hexamethyl-5-dodecanoylgroup and the like, and divalent groups induced therefrom.

In the “organic group having an alkyl group having not less than 10 andnot more than 300 carbon atoms and/or an alkenyl group having not lessthan 10 and not more than 300 carbon atoms”, the moiety other than the“alkyl group having not less than 10 and not more than 300 carbon atomsand/or alkenyl group having not less than 10 and not more than 300carbon atoms” can be determined freely. For example, it optionally has amoiety such as —O—, —S—, —COO—, —OCONH—, and —CONH—, and a hydrocarbongroup (monovalent group or divalent group) and the like as a linker.Examples of the “hydrocarbon group” include an aliphatic hydrocarbongroup, an aromatic aliphatic hydrocarbon group, a monocyclic saturatedhydrocarbon group, an aromatic hydrocarbon group and the like.Specifically, for example, monovalent groups such as an alkyl group, analkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, anaralkyl group and the like, and divalent groups derived therefrom areused. As “alkyl group”, “alkenyl group”, “alkynyl group”, “cycloalkylgroup”, “aryl group”, or “aralkyl group” as the moiety other than“aliphatic hydrocarbon group”, those similar to the aforementionedgroups can be mentioned. The “hydrocarbon group” is optionallysubstituted by a substituent selected from a halogen atom (chlorineatom, bromine atom, fluorine atom, iodine atom), a C₁₋₆ alkyl groupoptionally substituted by one or more halogen atoms, an oxo group andthe like.

The “organic group having an alkyl group having not less than 10 and notmore than 300 carbon atoms and/or an alkenyl group having not less than10 and not more than 300 carbon atoms” indicated as “R5 (group)” and/or“R⁷ (group)” constituting Z in the above-mentioned formula (I) maycontain plural “alkyl groups having not less than 10 and not more than300 carbon atoms and/or alkenyl groups having hot less than 10 and notmore than 300 carbon atoms” due to branching and the like. When aplurality of “alkyl group having not less than 10 and not more than 300carbon atoms and/or alkenyl group having not less than 10 and not morethan 300 carbon atoms” is present in the “organic group having an alkylgroup having not less than 10 and not more than 300 carbon atoms and/oran alkenyl group having not less than 10 and not more than 300 carbonatoms”, they may be the same or different.

The lower limit of the total carbon number of the “organic group havingan alkyl group having not less than 10 and not more than 300 carbonatoms and/or an alkenyl group having not less than 10 and not more than300 carbon atoms” for “R⁵ (group)” and/or “R⁷ (group)” constituting Z inthe above-mentioned formula (II) is preferably 10 or more, morepreferably 12 or more, further preferably 14 or more, still morepreferably 18 or more, and particularly preferably 30 or more. On theother hand, the upper limit of the total carbon number of the “organicgroup having a an alkyl group having not less than 10 and not more than300 carbon atoms and/or an alkenyl group having not less than 10 and notmore than 300 carbon atoms” for “R⁵ (group)” and/or “R⁷ (group)” ispreferably 200 or less, more preferably 150 or less, further preferably120 or less, still more preferably 100 or less, especially preferably 80or less, and particularly preferably 60 or less. When the carbon numberis higher, the crystallinity or solubility of the compound of thepresent invention in a polar solvent is fine even when the morpholinooligonucleotide has a long chain.

A preferable embodiment of Z represented by the above-mentioned formula(a2) is a group represented by the formula (a2), wherein, in the formula(a2),

-   R_(a) and R_(b) are both hydrogen atoms;-   R⁴ is a hydrogen atom,-   Q in the number of k is —O—,-   R⁵ in the number of k are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms (e.g., C₁₀₋₄₀ alkyl group); and-   k is an integer of 1-3.

Another preferable embodiment of Z represented by the above-mentionedformula (a2) is a group wherein, in the formula (a2),

-   k is an integer of 1-3;-   R_(a) and R_(b) are both hydrogen atoms;-   R⁴ is a hydrogen atom,-   Q in the number of k is —O—,-   R⁵ in the number of k are each independently a benzyl group having    1-3 alkyl groups having not less than 10 and not more than 300    carbon atoms and/or alkenyl groups having not less than 10 and not    more than 300 carbon atoms, or a cyclohexyl group having 1-3 alkyl    groups having not less than 10 and not more than 300 carbon atoms    and/or alkenyl groups having not less than 10 and not more than 300    carbon atoms; and-   ring A optionally further has, in addition to QR⁵ in the number of    k, substituent(s) selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen atom,    and a C₁₋₆ alkoxy group optionally substituted by a halogen atom.

Another preferable embodiment of Z represented by the above-mentionedformula (a2) is a group wherein, in the formula (a2),

-   R_(a) is a hydrogen atom; and-   R_(b) is a group represented by the above-mentioned formula (a3)    (wherein * indicates a bonding position; j is an integer of 0 to 3;    Q in the number of j is —O—; R⁷ in the number of j are each    independently a C₁₀₋₄₀ alkyl group; and-   R⁴ and R⁶ are both hydrogen atoms.

A still another preferable embodiment of Z represented by theabove-mentioned formula (a2) is a group wherein, in the formula (a2),

-   R_(a) is a hydrogen atom;-   R_(b) is a group represented by the above-mentioned formula (a3)    (wherein * indicates a bonding position; j is an integer of 0 to 3;    Q in the number of j is —O—; R⁷ in the number of j are each    independently a C₁₀₋₄₀ alkyl group;-   R⁶ is joined with R⁴ of ring A to form a single bond or —O—, and    therefore, ring A and ring B form a fluorenyl group or a xanthenyl    group in combination.

Another preferable embodiment of Z represented by the above-mentionedformula (a2) is a group wherein, in the formula (a2).

-   R_(a) and R_(b) are joined to form an oxygen atom;-   R⁴ is a hydrogen atom,-   Q in the number of k is —O—,-   R⁵ in the number of k are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms (e.g., C₁₀₋₄₀ alkyl group); and-   k is an integer of 1-3.

Another preferable embodiment of Z represented by the above-mentionedformula (a2) is a group wherein, in the formula (a2),

-   k is an integer of 1-3;-   R_(a) and R_(b) are joined to form an oxygen atom;-   R⁴ is a hydrogen atom;-   Q in the number of k is —O—,-   R⁵ in the number of k are each independently a benzyl group having    1-3 alkyl groups having not less than 10 and not more than 300    carbon atoms and/or alkenyl groups having not less than 10 and not    more than 300 carbon atoms, or a cyclohexyl group having 1-3 alkyl    groups having not less than 10 and not more than 300 carbon atoms    and/or alkenyl groups having not less than 10 and not more than 300    carbon atoms; and-   ring A optionally further has, in addition to QR⁵ in the number of    k, substituent(s) selected from the group consisting of a halogen    atom, a C₁₋₆ alkyl group optionally substituted by a halogen    atom(s), and a C₁₋₆ alkoxy group optionally substituted by a halogen    atom(s).

As the protecting group represented by the formula (II): Z—Y-L-, a groupnot easily cleaved under acidic conditions under which the protectinggroup P¹ of the morpholine ring nitrogen atom at the 3′-terminus can beremoved, and cleaved under basic conditions is preferable.

Representative examples of the protecting group include a group wherein,for example,

-   L is a group represented by the above-mentioned formula (a1)    (preferably a succinyl group etc.), and-   Z—Y is the following group:-   a 3,4,5-tri(octadecyloxy)benzyloxy group,-   a 3,5-di(docosyloxy)benzyloxy group,-   a 3,5-bis[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzyloxy group,-   a 3,4,5-tris[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzyloxy group,-   a 3,4,5-tri(octadecyloxy)benzylamino group,-   a 2,4-di(docosyloxy)benzylamino group,-   a 3,5-di(docosyloxy)benzylamino group,-   a di(4-docosyloxyphenyl)methylamino group,-   a 4-methoxy-2-[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzylamino    group,-   a    4-methoxy-2-[3′,4′,5′-tri(octadecyloxy)cyclohexylmethyloxy]benzylamino    group,-   a 2,4-di(dodecyloxy)benzylamino group,-   a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,-   a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group,-   a 3,5-bis[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzylamino group, or-   a 3,4,5-tris[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzylamino group.

As another embodiment of the protecting group Z—Y-L-, the followingbenzylsuccinyl groups and diphenylmethylsuccinyl groups can bementioned.

-   a 2-{2,4-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a 3,5-di(2′,3′-dihydrophytyloxy)benzylsuccinyl group;-   a 4-(2′,3′-dihydrophytyloxy)benzylsuccinyl group;-   a    2-{1-[(2-chloro-5-(2′,3′-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl    group;-   a 3,4,5-tri(2′,3′-dihydrophytyloxy)benzylsuccinyl group;-   a    2-{3,4,5-tri(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a 2-{4-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{2-(3′,4′,5′-tri(2″,3″-dihydrophytyloxy)benzyloxy]-4-methoxybenzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{4-(2′,3′-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl    group;-   a 4-(2′,3′-dihydrophytyloxy)-2-methylbenzylsuccinyl group;-   a    2-{4-(2′,3′-dihydrophytyloxy)-2-methylbenzylaminocarbonyl}ethylcarbonyl    group;-   a 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl    group;-   a    2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl    group;-   a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group;-   a 2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a 2-{3,5-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{1-[2,3,4-tri(2′,3′-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl    group;-   a    2-{1-[4-(2′,3′-dihydrophytyloxy)phenyl]-4′-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;-   a 3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylsuccinyl    group; and-   a    2-{3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl    group.

Another embodiment of the protecting group represented by the formula(II): Z—Y-L- is a group wherein

-   L and Y are each a single bond,-   Z shows the formula (a2),-   R_(a) and R_(b) are joined to form an oxygen atom;-   R⁴ is a hydrogen atom,-   Q in the number of k is —O—,-   R⁵ in the number of k are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms (e.g., C₁₀₋₄₀) alkyl group); and-   k is an integer of 1-3.

Another embodiment of the protecting group represented by the formula(II): Z—Y-L- is a group wherein

-   L shows the formula (a1),-   L₂ is ***N(R³)—R¹—N(R²)C(═O) ** (wherein ** indicates the bonding    position to L¹, *** indicates the bonding position to Y, R¹ is an    optionally substituted C₁₋₂₂ alkylene group, R² and R³ are each    independently a hydrogen atom or an optionally substituted C₁₋₂₂    alkyl group, or R² and R³ are optionally joined to form an    optionally substituted C₁₋₂₂ alkylene bond),-   Y is a single bond,-   Z shows the formula (a2),-   R_(a) and R_(b) are joined to form an oxygen atom;-   R⁴ is a hydrogen atom,-   Q in the number of k is —O—,-   R⁵ in the number of k are each independently an organic group having    an alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms (e.g., C₁₀₋₄₀ alkyl group); and-   k is an integer of 1-3.

A preferable embodiment of the compound of the present inventionrepresented by the formula (I) is a compound of the formula (I), wherein

-   m is 0,-   Base is a cytosyl group, a uracil group, a thyminyl group, an adenyl    group, or a guanyl group, each of which is optionally protected;-   P¹ is a trityl group, a di(C₁₋₆ alkoxy)trityl group or a mono(C₁₋₆    alkoxy)trityl group;-   S¹ is a single bond; and-   Z—Y-L is the combination of each group shown as a preferable    embodiment in the aforementioned formula (I).

Another preferable embodiment of the compound of the present inventionrepresented by the formula (I) is a compound of the formula (I), wherein

-   m is 0,-   Base is a cytosyl group, a uracil group, a thyminyl group, an adenyl    group, or a guanyl group, each of which is optionally protected;-   P¹ is a trityl group, a dimethoxytrityl group or a monomethoxytrityl    group;-   S¹ is a single bond; and-   Z—Y-L is the combination of each group shown as a preferable    embodiment in the aforementioned formula (I).

A still another preferable embodiment of the compound of the presentinvention represented by the formula (I) is a compound of the formula(I), wherein

-   m is 0,-   Base is a cytosyl group, a uracil group, a thyminyl group, an adenyl    group, or a guanyl group, each of which is optionally protected;-   P¹ is a trityl group;-   S¹ is a single bond; and-   Z—Y-L is the combination of each group shown as a preferable    embodiment in the aforementioned formula (I).

[Production Method of the Compound of the Present Invention]

A production method of the compound of the present invention representedby the formula (I) wherein m is 0, and S¹ is a single bond (hereinafterto be referred to as “the formula (Ia)”) is not particularly limited,and it can be produced by a method known per se (Richard T. Pon et al.,Nucleic Acids Research 2004, 32, 623-631) or a method analogous thereto.

A general production method of a compound of the above-mentioned formula(Ia) wherein L is a succinyl group is shown below.

(wherein each symbol is as defined above.)

Morpholino nucleoside (a) wherein the 3′-terminus morpholine ringnitrogen atom is protected by a protecting group P¹ is reacted withsuccinic anhydride in the presence of a base to give compound (b)wherein succinic acid is introduced into the 5′-hydroxy group. Compound(b) is subjected to a dehydration condensation with a precursor (Z—Y—H)(alcohol or amine) of the protecting group in the presence of acondensing agent, whereby a compound represented by the formula (Ia) canbe obtained.

The conversion step of the above-mentioned morpholino nucleoside (a) tocompound (b) is advantageously performed in a solvent inert to thereaction. While such solvent is not particularly limited as long as thereaction proceeds, halogenated hydrocarbon solvents such asdichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachlorideand the like, aromatic hydrocarbon solvents such as benzene, toluene,xylene and the like, aliphatic hydrocarbon solvents such as pentane,hexane, heptane, octane and the like, ether solvents such as diethylether, tetrahydrofuran, cyclopentyl methyl ether and the like, and mixedsolvents thereof are preferable. Of these, dichloromethane andchloroform are particularly preferable.

While the base is not particularly limited, for example, an organic basementioned below can be used, with preference given toN,N-dimethylaminopyridine, triethylamine and the like.

The above-mentioned dehydration condensation step is advantageouslyperformed in a solvent inert to the reaction. While such solvent is notparticularly limited as long as the reaction proceeds, halogenatedhydrocarbon solvents such, as dichloromethane, 1,2-dichloroethane,chloroform, carbon tetrachloride and the like, aromatic hydrocarbonsolvents such as benzene, toluene, xylene and the like, or aliphatichydrocarbon solvents such as pentane, hexane, heptane, octane and thelike, and mixed solvents thereof are preferable. Of these,dichloromethane and chloroform are particularly preferable.

Examples of the condensing agent used for the condensation reaction ofcompound (b) with Z—Y—H include dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIC),N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and hydrochloride thereof(EDC HCl), (benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBop),O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU),1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxidehexafluorophosphate (HCTU), O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and the like. Of these,HBTU, HCTU, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide andhydrochloride thereof (EDC HCl) are preferable.

The amount of the condensing agent to be used is 1 to 10 mol, preferably1 to 5 mol, per 1 mol of compound (b). The amount of Z—Y—H to be used is1 to 10 mol, preferably 1 to 5 mol, per 1 mol of compound (b). While thereaction temperature is not particularly limited as long as the reactionproceeds, it is preferably −10° C. to 50° C., more preferably 0° C. to30° C. The reaction time is 30 min to 70 hr.

A compound of the above-mentioned formula (Ia) wherein L is other than asuccinyl group can also be produced by performing a reaction similar tothe above-mentioned production method except that a corresponding acidanhydride, a corresponding dicarboxylic acid halide, an activated esterof corresponding dicarboxylic acid and the like is used instead ofsuccinic anhydride.

A compound wherein S¹ is “O—S²** (wherein each symbol is as definedabove) can be produced by introducing the aforementioned “substituenthaving a hydroxy group” into the 5′-hydroxy group of the morpholinonucleoside (a) by a known method (e.g., the method described in WO2008/008113) and thereafter following the above-mentioned method.

A compound wherein Y is a single bond can be produced by reacting anactivated derivative (halide, acid halide, activated carboxy group etc.)of Z—Y—H with morpholino nucleoside (a) by a method known per se, orreacting Z—Y—H with morpholino nucleoside (a) in the presence of acondensing agent. The condensation reaction of Z—Y—H and morpholinonucleoside (a) can be performed in the same manner as in thecondensation reaction of Z—Y—H and compound (b).

A compound of the above-mentioned formula (I) wherein m is one or morecan be produced by repeating the 5′-terminus elongation processaccording to the following production method of the present inventionand using a compound represented by the formula (Ia) as a startingmaterial.

While the production method of precursor (Z—Y—H) (alcohol, amine orcarboxylic acid) of the aforementioned protecting group is notparticularly limited, it can be produced from a starting compoundaccording to a method known per se (e.g., Bull. Chem. Soc. Jpn. 2001,74, 733-738, JP-A-2000-44493, WO 2006/104166, WO 2007/034812, WO2007/122847, WO 2010/113939, JP-A-2010-275254, WO 2012/157723 etc.) or amethod analogous thereto.

A compound to be used as a starting compound, for example, a halidecorresponding to R⁵ and R⁷ constituting Z in the formula (I) and thelike is a commercially available product, or can be produced accordingto a method known per se or a method analogous thereto.

The precursor (Z—Y—H) of the protecting group can be produced by amethod known per se or a method analogous thereto, as mentioned above.When a starting compound has a substituent (e.g., hydroxy group, aminogroup, carboxy group) that influences the reaction, the startingcompound is generally protected in advance by a suitable protectinggroup according to a known method and then subjected to the reaction.Such protecting group can be removed after the reaction by a knownmethod such as an acid treatment, an alkali treatment, a catalyticreduction and the like.

While the production method of morpholino nucleoside (a) wherein the3′-terminus morpholine ring nitrogen atom is protected by protectinggroup P¹ is not particularly limited, it can be produced from morpholinonucleoside (1) by a method known per se (e.g., see WO 91/09033A1) or amethod analogous thereto.

For example, when P¹ is a trityl group, morpholino nucleoside (1) isreacted with trityl chloride in the presence of a base such astriethylamine and the like, whereby compound (a) can be obtained.

Compound (a) wherein P¹ is a hydrogen atom can be obtained by subjectingcompound (a) wherein P¹ is a temporary protecting group to thebelow-mentioned deprotection step (1′).

[Production Method of the Present Invention]

The production method of the morpholino oligonucleotide of the presentinvention (hereinafter to be also referred to as the “production methodof the present invention”) is explained. Specifically, a productionmethod from appropriately protected n-mer morpholino oligonucleotide toappropriately protected n+p-mer morpholino oligonucleotide is explained.For example, when n=1, n-mer morpholino oligonucleotide is to beunderstood as “morpholino nucleoside”, and when p=1, p-mer morpholinooligonucleotide is to be understood as “morpholino nucleoside” andn+p-mer morpholino oligonucleotide is to be understood as“dinucleotide”.

The production method of the present invention preferably comprises thefollowing step (1).

-   (1) A step of obtaining n+p-mer morpholino oligonucleotide,    comprising condensing a p-mer morpholino oligonucleotide (p is any    integer of one or more) wherein a 5′-hydroxy group is activated    (thio)phosphated or activated (thio)phosphoramidated, and a    morpholine ring nitrogen atom is protected by a temporary protecting    group removable under acidic conditions (hereinafter sometimes to be    simply referred to as “activated morpholino nucleotide”), with an    n-mer morpholino oligonucleotide (n is an integer of one or more)    wherein a 5′-hydroxy group or, when the 5′-hydroxy group has a    substituent having a hydroxy group, the hydroxy group present on the    substituent is protected by a protecting group having an alkyl group    having not less than 10 and not more than 300 carbon atoms and/or an    alkenyl group having not less than 10 and not more than 300 carbon    atoms, and the morpholine ring nitrogen atom is not protected, by a    (thio)phosphoramidate bond or (thio)phosphorodiamidate bond via the    morpholine ring nitrogen atom.

While the upper limit of n is not particularly limited, it is preferablynot more than 50, more preferably not more than 30, further preferablynot more than 20.

While the upper limit of p is not particularly limited, it is preferablynot more than 50, more preferably not more than 30, further preferablynot more than 20, still further preferably not more than 5, particularlypreferably not more than 3.

The production method of the present invention preferably furthercontains the following step (1′), whereby n-mer morpholinooligonucleotide wherein the 5′-hydroxy group or the hydroxy grouppresent on the substituent of the 5′-hydroxy group used in step (1) isprotected by a protecting group having an alkyl group having not lessthan 10 and not more than 300 carbon atoms and/or an alkenyl grouphaving not less than 10 and not more than 300 carbon atoms, and themorpholine ring nitrogen atom is not protected is prepared.

-   (1′) A step of removing the temporary protecting group of the    morpholine ring nitrogen atom by reacting, before the condensation    step (1), the n-mer morpholino oligonucleotide wherein the    5′-hydroxy group or the hydroxy group present on the substituent of    the 5′-hydroxy group is protected by a protecting group having an    alkyl group having not less than 10 and not more than 300 carbon    atoms and/or an alkenyl group having not less than 10 and not more    than 300 carbon atoms, and the morpholine ring nitrogen atom is    protected by a temporary protecting group removable under acidic    conditions, with an acid in a non-polar solvent.

Preferably, step (1′) further includes a step of removing the temporaryprotecting group of the morpholine ring nitrogen atom and thereafterneutralizing same with an organic base. Consequently, steps (1′) and (1)can be continuously performed in a liquid and morpholino oligonucleotidecontaining elongated morpholino nucleotide can be continuously obtained.

Since the 5′-hydroxy group or the hydroxy group present on thesubstituent of the 5′-hydroxy group of the n-mer morpholinooligonucleotide is protected by a protecting group having an alkyl grouphaving not less than 10 and not more than 300 carbon atoms and/or analkenyl group having not less than 10 and not more than 300 carbonatoms, the liposolubility of the obtained n+p-mer morpholinooligonucleotide is improved and, for example, by comprising thefollowing step (2), the n+p-mer morpholino oligonucleotide can bepurified conveniently and effectively by removing excess startingmaterials and by-products:

-   (2) a step of adding a polar solvent to the reaction mixture    obtained in step (1′) or (1) to precipitate n+p-mer oligonucleotide,    and obtaining same by solid-liquid separation.

Step (2) may be performed, as shown in the following (A)-(C), with boththe reaction mixtures of steps (1′) and (1), or only one of the reactionmixtures of steps (1′) and (1).

-   (A) step (1′)→step (2)→step (1)→step (2)-   (B) step (1′)→step (1)→step (2)-   (C) step (1′)→step (2)→step (1)

A protecting group having an alkyl group having not less than 10 and notmore than 300 carbon atoms and/or an alkenyl group having not less than10 and not more than 300 carbon atoms is preferably the formula (II)Z—Y-L- (wherein each symbol is as described above), in which case theabove-mentioned step (2) can be more efficiently performed.

When the amount of the by-product generated can be controlled by themanagement of equivalent of the starting materials and controlling thereaction, it is preferable to repeat steps (1′) and (1) as a basic unit,which includes step (2).

Since the generation of by-product can be strictly managed and controlled and highly pure morpholino oligonucleotide can be obtained, it ispreferable to repeat any of the above-mentioned (A)-(C) containing step(1′) to step (2) as a basic unit.

Morpholino oligonucleotide can be isolated and produced by furtherincluding step (3) in the production method of the present invention:

-   (3) A step of removing all the protecting groups of the obtained    n+p-mer morpholino oligonucleotide. Each step is explained in detail    in the following.    1. Explanation of “n-mer Morpholino Oligonucleotide”

First, n-mer morpholino oligonucleotide used as a starting material ofsteps (1′) and (1) is explained.

The n-mer morpholino oligonucleotide used in step (1′) is, for example,n-mer morpholino oligonucleotide wherein the 5′-hydroxy group or thehydroxy group present on the substituent of the 5′-hydroxy group isprotected by a protecting group having an alkyl group having not lessthan 10 and not more than 300 carbon atoms and/or an alkenyl grouphaving not less than 10 and not more than 300 carbon atoms, and themorpholine ring nitrogen atom is protected by a temporary protectinggroup removable under acidic conditions, as shown by, for example, thefollowing formula (i), and the n-mer morpholino oligonucleotide used instep (1) is, for example, n-mer morpholino oligonucleotide wherein the5′-hydroxy group or the hydroxy group present on the substituent of the5′-hydroxy group is protected by a protecting group having an alkylgroup having not less than 10 and not more than 300 carbon atoms and/oran alkenyl group having not less than 10 and not more than 300 carbonatoms, and the morpholine ring nitrogen atom is not protected, as shownby, for example, the following formula (ii).

-   (wherein-   m is any integer of not less than 0 which corresponds to n-1,-   P¹′ is a temporary protecting group removable under acidic    conditions,-   P² is a protecting group having an alkyl group having not less than    10 and not more than 300 carbon atoms and/or an alkenyl group having    not less than 10 and not more than 300 carbon atoms, and-   other symbols are the same as respective definitions in the formula    (I).)

Each symbol in the formulas (i) and (ii) is explained below.

While the upper limit of m is not particularly limited, it is generallynot more than 99, preferably not more than 74, more preferably not morethan 49, further preferably not more than 29.

The temporary protecting group removable under acidic conditions for P¹′in the formula (i) is not particularly limited as long as it can bedeprotected under acidic conditions and can be used as ahydroxy-protecting group. Examples thereof include a trityl group, a9-(9-phenyl)xanthenyl group, a 9-phenylthioxanthenyl group, di(C₁₋₆alkoxy)trityl groups such as a 1,1-bis(4-methoxyphenyl)-1-phenylmethylgroup, a dimethoxytrityl group and the like, mono(C₁₋₁₈ alkoxy)tritylgroups such as 1-(4-methoxyphenyl)-1,1-diphenylmethyl group,monomethoxytrityl group and the like, and the like can be mentioned.Among these, a trityl group, a monomethoxytrityl group and adimethoxytrityl group are preferable, and a trityl group and adimethoxytrityl group is more preferable, in view of easiness ofdeprotection and easy availability.

The “protecting group having an alkyl group having not less than 10 andnot more than 300 carbon atoms and/or an alkenyl group having not lessthan 10 and not more than 300 carbon atoms” for P² in the formulas (i)and (ii) is not particularly limited as long as it is a group stableunder acidic conditions under which the protecting group of the3′-terminus morpholine ring nitrogen atom can be removed and candissolve n-mer morpholino oligonucleotide in a non-polar solvent as areaction solvent to allow for the progress of the reactions in steps(1′) and (1). A group represented by the following formula (II) ispreferable.

Z—Y-L-   (II)

[wherein each symbol is as defined above].

When a protecting group having an alkyl group having not less than 10and not more than 300 carbon atoms and/or an alkenyl group having notless than 10 and not more than 300 carbon atoms, more preferably, agroup represented by the formula (II), is used as a hydroxy-protectinggroup of the 5′-hydroxy group or the hydroxyl group present on thesubstituent of the 5′-hydroxy group, the liposolubility and solubilityof n-mer morpholino oligonucleotide and n+p-mer morpholinooligonucleotide in a solvent (particularly, non-polar solvent) can beimproved, steps (1′) and (1) can be performed smoothly and, as in thebelow-mentioned step (2), n-mer morpholino oligonucleotide or n+p-mermorpholino oligonucleotide can be isolated and purified conveniently.

Preferable embodiments of L, Y and Z in the formula (II) are the same asthose of L, Y and Z in a group represented by the formula (I).

2. Explanation of “p-mer Morpholino Oligonucleotide”

First, p-mer morpholino oligonucleotide used as a starting material ofstep (1) is explained.

The “p-mer morpholino oligonucleotide (p is any integer of one or more)wherein a 5′-hydroxy group is activated (thio)phosphated or activated(thio)phosphoramidated, and a morpholine ring nitrogen atom is protectedby a temporary protecting group removable under acidic conditions” usedin step (1) is not particularly limited as long as the structuralrequirements are met.

The “5′-hydroxy group is activated (thio)phosphated or activated(thio)phosphoramidated” means that 5′-hydroxy group of the morpholinooligonucleotide is, for example, modified by a group represented by thefollowing formula (c):

-   (wherein-   * indicates the bonding position to the 5′-terminus hydroxyl group    of morpholino oligonucleotide,-   L¹ is a leaving group,-   X is a C₁₋₆ alkoxy group, a di-C₁₋₆ alkylamino group, or a    piperazino group wherein the 4-position nitrogen atom is protected    by a protecting group, and optionally further substituted, and-   W is an oxygen atom or a sulfur atom).

The “activated phosphated” means the above-mentioned formula wherein Xis a C₁₋₆ alkoxy group, and W is an oxygen atom.

The “activated thiophosphated” means the above-mentioned formula whereinX is a C₁₋₆ alkoxy group, and W is a sulfur atom.

The “activated phosphoramidated” means the above-mentioned formulawherein X is a di-C₁₋₆ alkylamino group or a piperazino group whereinthe 4-position nitrogen atom is protected by a protecting group, andoptionally further substituted, and W is an oxygen atom.

The “activated (thio)phosphoramidated” means the above-mentioned formulawherein X is a di-C₁₋₆ alkylamino group or a piperazino group whereinthe 4-position nitrogen atom is protected by a protecting group, andoptionally further substituted, and W is a sulfur atom.

Examples of the leaving group for L¹ include a halogen atom, amethanesulfonyloxy group, a p-toluenesulfonyloxy group and the like, anda chlorine atom is preferable.

The definitions, examples and preferable embodiments of X and W are asexplained for the above-mentioned formula (I).

The definitions, examples and preferable embodiments of the “temporaryprotecting group removable under acidic conditions” are as explained forthe above-mentioned formula (I).

As a preferable p-mer morpholino oligonucleotide used in step (1), acompound represented by the formula (iii) can be mentioned.

-   (wherein-   q is any integer of not less than 0 which corresponds to p-1,-   P¹″ is a temporary protecting group removable under acidic    conditions, and-   other symbols are the same as respective definitions in the    formula (I) and the formula (c)).

q in the formula (iii) is preferably 0. While the upper limit of q isnot particularly limited, it is generally not more than 99, preferablynot more than 74, more preferably not more than 49, further preferablynot more than 29.

The temporary protecting group removable under acidic conditions for P¹″in the formula (iii) is not particularly limited as long as it can bedeprotected under acidic conditions and can be used as ahydroxy-protecting group. Examples thereof include a trityl group, a9-(9-phenyl)xanthenyl group, a 9-phenylthioxanthenyl group, di(C₁₋₆alkoxy) trityl groups such as a 1,1-bis(4-methoxyphenyl)-1-phenylmethylgroup, dimethoxytrityl and the like, mono(C₁₋₁₈ alkoxy)trityl groupssuch as 1-(4-methoxyphenyl)-1,1-diphenylmethyl group, monomethoxytritylgroup and the like, and the like can be mentioned. Among these, a tritylgroup, a monomethoxytrityl group and a dimethoxytrityl group arepreferable, and a trityl group and a dimethoxytrityl group are morepreferable, in view of easiness of deprotection and easy availability.

Preferable embodiments of other symbols in the formula (iii) are asexplained for the above-mentioned formulas (I) and (c).

The p-mer morpholino oligonucleotide of the present invention can beprepared by a method known per se (e.g., the method described in WO91/09033A1), or a method analogous thereto. For example, a compoundwherein L¹ is a chlorine atom can be produced by reacting a compound ofthe following formula (iii′) which is a compound represented by theformula (iii), wherein the 5′-hydroxy group is not activated, with, forexample, dichloro(thio)phosphate or dichloro (thio)phosphoramidaterepresented by the formula (d): Cl₂P(═W)(X) (wherein W and X are asdefined above).

As dichloro(thio)phosphate or dichloro(thio)phosphoramidate representedby the formula (d), a commercially available product can be used, or canbe produced by a known method (e.g., the methods described in WO91/09033, WO 2008/008113 etc.) or a method analogous thereto.

A compound of the formula (iii′) can be prepared by a known method, forexample, WO91/09033 and the like.

3. Explanation of Steps (1′)-(3)

While steps (1′)-(3) are explained below by reference to the formulas(i), (ii), (iii) and the like for convenience, they are not limitedthereby.

Step (1′) (Deprotection Step)

This step is a step of removing the temporary protecting group of themorpholine ring nitrogen atom by reacting, before the condensation step(1), n-mer morpholino oligonucleotide (i) wherein the 5′-hydroxy groupor the hydroxy group present on the substituent of the 5′-hydroxy groupis protected by a protecting group having an alkyl group having not lessthan 10 and not more than 300 carbon atoms and/or an alkenyl grouphaving not less than 10 and not more than 300 carbon atoms, and themorpholine ring nitrogen atom is protected by a temporary protectinggroup removable under acidic conditions, with an acid in a non-polarsolvent to give n-mer morpholino oligonucleotide (ii) wherein the5′-hydroxy group or the hydroxy group present on the substituent of the5′-hydroxy group is protected by a protecting group having an alkylgroup having not less than 10 and not more than 300 carbon atoms and/oran alkenyl group having not less than 10 and not more than 300 carbonatoms, and morpholine ring nitrogen atom is not protected.

(wherein each symbol is as defined above).

This step is performed in a solvent that does not influence thereaction. Since a higher solubility of the solvent is expected to affordsuperior reactivity, a non-polar solvent showing high solubility ofn-mer morpholino oligonucleotide (1) of the present invention ispreferably selected. Specifically, examples thereof include halogenatedsolvents such as chloroform, dichloromethane, 1,2-dichloroethane and thelike; aromatic solvents such as benzene, toluene, xylene, mesitylene andthe like; ester solvents such as ethyl acetate, isopropyl acetate andthe like; aliphatic solvents such as hexane, pentane, heptane, octane,nonane, cyclohexane and the like; non-polar ether solvents such asdiethyl ether, cyclopentyl methyl ether, tert-butyl methyl ether and thelike. Among them, dichloromethane, chloroform, 1,2-dichloroethane,benzene, toluene, xylene, mesitylene, hexane, pentane, heptane, nonane,cyclohexane, ethyl acetate, isopropyl acetate, tert-butyl methyl ether,cyclopentyl methyl ether, and the like are preferable. Two or more kindsof these solvents may be used in a mixture in an appropriate ratio.

In this step, the concentration of n-mer morpholino oligonucleotide (i)in a solvent is not particularly limited as long as the oligonucleotideis dissolved, it is preferably 1 to 30 mass %.

While the acid to be used in this step is not particularly limited aslong as good deprotection can be achieved, trifluoroacetic acid,cyanopyridine trifluoroacetate: and trifluoroethanol, trimethylaminetrifluoroacetate, cyanoacetic acid, acetic acid, dichloroacetic acid,phosphoric acid, mesylic acid, tosic acid, hydrochloric acid and thelike are preferably used.

Since good reaction can be achieved, trifluoroacetic acid, cyanopyridinetrifluoroacetate, trimethylamine trifluoroacetate, and cyanoacetic acidare more preferable, cyanopyridine trifluoroacetate and trimethylaminetrifluoroacetate are further preferable, and trimethylaminetrifluoroacetate is particularly preferable. These acids may be dilutedwith the above-mentioned non-polar solvent. When the aforementioned acidis used, it may be combined with a particular base (e.g., triethylamineetc.) to appropriately adjust the acidity before use.

The amount of the acid to be used in this step is 1 to 100 mol,preferably 1 to 40 mol, per 1 mol of n-mer morpholino oligonucleotide(i).

In this step, a cation scavenger may be added to prevent side reactionsdue to cationized compound and the like of protecting group P¹ such astrityl cation and the like resulting from deprotection. Examples ofpreferable cation scavengers include pyrrole, indole, ethanol,2,2,2-trifluoroethanol, methanol, anisole, p-cresol, triisopropylsilane,mercaptoethanol, thioanisole and the like. Among these, pyrrole, indole,ethanol, and 2,2,2-trifluoroethanol are more preferable, and ethanol and2,2,2-trifluoroethanol are particularly preferable.

The amount of the cation scavenger to be used can be appropriatelydetermined in consideration of an excess amount of p-mer morpholinooligonucleotide (iii) relative to n-mer morpholino oligonucleotide (ii)(number of moles of p-mer morpholino oligonucleotide (iii)—number ofmoles of n-mer morpholino oligonucleotide (ii)), and is preferably 1-20equivalents, more preferably 1-10 equivalents, relative to the excessamount (moles).

While the reaction temperature in this step is not particularly limitedas long as the reaction proceeds, it is preferably −10° C. to 50° C.,more preferably 0° C. to 40° C. While the reaction time varies dependingon the kind of n-mer morpholino oligonucleotide to be used, the kind ofacid, the kind of solvent, the reaction temperature and the like, it is5 min to 5 hr.

When an acid used as a deprotecting agent is present in the condensationstep of the next step, deprotection of protecting group P¹″ of p-mermorpholino oligonucleotide (iii) is induced. Therefore, a removaltreatment or a neutralization treatment is necessary. To continuouslyperform the deprotection step and subsequent condensation step in asolution, it is preferable to remove the temporary protecting group ofthe 3′-terminus morpholine ring nitrogen atom and neutralize thecompound with an organic base in this step.

The organic base to be used for neutralization is not particularlylimited as long as it can neutralize the above-mentioned acids, and theobtained salt can function as a condensing agent. Since the reactionproceeds smoothly, N,N-diisopropylethylamine, pyridine, 4-cyanopyridine,triethylamine is preferable, N,N-diisopropylethylamine and triethylamineare more preferable, and N,N-diisopropylethylamine is particularlypreferable.

The amount of the organic base to be used in this step is 1 to 10 mol,preferably 1 to 3 mol, per 1 mol of the acid.

A particularly preferable combination of an acid and an organic base inthis step is that of cyanopyridine trifluoroacetate andN,N-diisopropylethylamine or cyanoacetic acid andN,N-diisopropylethylamine.

Step (1) (Condensation Step)

In this step, a p-mer morpholino oligonucleotide (iii) wherein a5′-hydroxy group is activated (thio)phosphated or activated(thio)phosphoramidated, and a morpholine ring nitrogen atom is protectedby a temporary protecting group removable under acidic conditions iscondensed with an n-mer morpholino oligonucleotide (ii) wherein a5′-hydroxy group or a hydroxy group present on a substituent of the5′-hydroxy group is protected by a protecting group having an alkylgroup having not less than 10 and not more than 300 carbon atoms and/oran alkenyl group having not less than 10 and not more than 300 carbonatoms, and the morpholine ring nitrogen atom is not protected, by a(thio)phosphoramidate bond, or (thio)phosphorodiamidate bond via themorpholine ring nitrogen atom to give n+p-mer morpholino oligonucleotide(iv).

(wherein each symbol is as defined above).

As the p-mer morpholino oligonucleotide (iii) wherein a 5′-hydroxy groupis activated (thio)phosphated or activated (thio)phosphoramidated, and amorpholine ring nitrogen atom is protected by a temporary protectinggroup removable under acidic conditions, a morpholino nucleoside whereinp is 1 (i.e., morpholino nucleoside wherein 5′-hydroxy group isactivated (thio)phosphated or activated (thio)phosphoramidated, andmorpholine ring nitrogen atom is protected by temporary protecting groupP¹″) is preferable.

In this step, the n-mer morpholino oligonucleotide (ii) to be used isnot particularly limited, and one obtained in the aforementioned step(1′) can be preferably used. In this case, a p-mer morpholinooligonucleotide (iii) only needs to be added directly to the reactionmixture after step (1′), without isolating the n-mer morpholinooligonucleotide (ii).

Alternatively, after step (1′), the reaction mixture may be subjected tothe below-mentioned step (2), n-mer morpholino oligonucleotide (ii) isonce isolated and dissolved in a given solvent, and thereafter, p-mermorpholino oligonucleotide (iii) may be added.

This step is performed in a solvent that does not influence thereaction. A non-polar solvent showing high solubility of n-mermorpholino oligonucleotide (ii) of the present invention is preferablyselected. Specifically, examples thereof include halogenated solventssuch as chloroform, dichloromethane, 1,2-dichloroethane and the like;aromatic solvents such as benzene, toluene, xylene, mesitylene and thelike; ester solvents such as ethyl acetate, isopropyl acetate and thelike; aliphatic solvents such as hexane, pentane, heptane, octane,nonane, cyclohexane and the like; non-polar ether solvents such asdiethyl ether, cyclopentyl methyl ether, tert-butyl methyl ether and thelike. Among them, dichloromethane, chloroform, 1,2-dichloroethane,benzene, toluene, xylene, mesitylene, hexane, pentane, heptane, nonane,cyclohexane, ethyl acetate, isopropyl acetate, tert-butyl methyl ether,cyclopentyl methyl ether, and the like are preferable. Two or more kindsof these solvents may be used in a mixture in an appropriate ratio. Inaddition, a polar solvent may be mixed at an appropriately ratio as longas n-mer morpholino oligonucleotide (ii) is dissolved. Specifically,polar solvents such as nitrile solvents such as acetonitrile,propionitrile and the like; ketone solvents such as acetone, 2-butanoneand the like; polar ether solvents such as 1,4-dioxane, tetrahydrofuranand the like; amide solvents such as N,N-dimethylformamide,N,N-dimethylacetamide, N-methylpyrrolidone, 1,3-dimethyl-2-imidazolinoneand the like, sulfoxide solvents such as dimethyl sulfoxide and the likecan be mentioned.

The amount of p-mer morpholino oligonucleotide (iii) to be used is 1-10mol, preferably 1-5 mol, more preferably 1-2 mol, per 1 mol of n-mermorpholino oligonucleotide (ii).

While the reaction temperature is not particularly limited as long asthe reaction proceeds, 0° C. to 100° C. is preferable, and 20° C. to 50°C. is more preferable. While the reaction time varies depending on thekind of n-mer morpholino oligonucleotide (ii) and p-mer morpholinooligonucleotide (iii) to be condensed, the reaction temperature and thelike, it is 5 min to 24 hr.

After the completion of the condensation reaction, the reaction mixtureis preferably treated with a quenching agent. Using a quenching agent,p-mer morpholino oligonucleotide (iii) remaining in the condensationreaction can be completely quenched, and induction of double addition inthe condensation reaction of the next cycle by the residual activatedmorpholino nucleotide can be avoided, which in turn prevents degradationof the quality of the object morpholino oligonucleotide.

The double addition refers to a doubly addition of the same residue ofactivated morpholino nucleotide used and remained in the condensationreaction of the previous cycle, which reacts, in the condensationreaction of the subsequent cycle.

As the quenching agent, any nucleophilic reagent can be used as long asit reacts with p-mer morpholino oligonucleotide (iii). Preferableexamples thereof include organic amine and thiol. Of them, secondaryamine is preferable, and morpholine is particularly preferable.

The amount of the quenching agent to be used can be appropriatelydetermined in consideration of an excess amount of p-mer morpholinooligonucleotide (iii) relative to n-mer morpholino oligonucleotide (ii)(number of moles of p-mer morpholino oligonucleotide (iii)—number ofmoles of n-mer morpholino oligonucleotide (ii)), and is preferably0.1-10 equivalents, more preferably 0.3-3 equivalents, relative to theexcess amount (moles).

After adding a quenching agent to the reaction mixture, p-mer morpholinooligonucleotide (iii) can be completely quenched by reacting the mixtureat 0° C.-100° C., preferably 20° C.-50° C., fox 30 min-24 hr, preferably30 min-5 hr.

Step (2) (Separation and Purification Step of MorpholinoOligonucleotide)

In n-mer morpholino oligonucleotide (ii) or n+p-mer morpholinooligonucleotide (iv) obtained in the above-mentioned step (1′) and/orstep (1), the hydroxy group at the 5′-terminus is protected by aprotecting group having an alkyl group containing not less than 10 andnot more than 300 carbon atoms, and/or an alkenyl group having not lessthan 10 and not more than 300 carbon atoms, preferably, a protectinggroup represented by the formula (II): Z—Y-L- (wherein each symbol is asdefined above). Therefore, very high liposolubility is imparted to themorpholino oligonucleotide, and the morpholino oligonucleotide can beconveniently isolated and purified by crystallization and extractionoperation alone, without requiring a complicated operation such ascolumn purification and the like.

In the following, an isolation and purification method of morpholinooligonucleotide by crystallization is explained, to which the presentinvention is not limited.

The isolation and purification step of morpholino oligonucleotide bycrystallization is a step of adding a polar solvent to the reactionmixture obtained in step (1′) or (1) to precipitate oligonucleotide, andobtaining same by solid-liquid separation.

A polar solvent may be directly added to the reaction mixture obtainedin step (1′) and/or step (1), or the reaction mixture obtained in step(1′) and/or step (1) may be concentrated and a polar solvent may beadded.

Examples of the polar solvent used to precipitate the object product,morpholino oligonucleotide, in this step include alcohol solvents suchas methanol, ethanol, isopropanol and the like; nitrile solvents such asacetonitrile, propionitrile and the like; ketone solvents such asacetone, 2-butanone and the like; amide solvents such asdimethylformamide, dimethylacetamide, N-methylpiperidone and the like,sulfoxide solvents such as dimethyl sulfoxide and the like; water etc.,and mixed solvent of two or more kinds thereof. A polar solvent aimingat increasing the precipitation efficiency can be used in the form of amixed solvent of an organic solvent and water. In this case, the contentof water in the polar solvent can be appropriately set to a preferablevalue depending on the organic solvent to be used. It is generally1-50%(V/V), preferably 10-30%(V/V). As the polar solvent, alcoholsolvent, nitrile solvent, and a mixed solvent of each of these solventsand water are preferable, methanol, acetonitrile, and a mixed solvent ofeach of these and water are more preferable, and acetonitrile and amixed solvent of acetonitrile and water are particularly preferable.

The production method of morpholino oligonucleotide of the presentinvention can afford the object morpholino oligonucleotide with highpurity and in a high yield by repeating the above-mentioned stepsdesired times in the order of (1′)-(2)-(1)-(2), (1′)-(2)-(1), or(1′)-(1)-(2).

Step (3) (Deprotection, Morpholino Oligonucleotide Isolation Step)

In the production method of morpholino oligonucleotide of the presentinvention, deprotection is performed after step (2) according to thekind and properties of the protecting group, whereby morpholinooligonucleotide is isolated. All protecting groups of the morpholinooligonucleotide can be removed according to the deprotection methoddescribed in Greene's PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th ed.,Wiley-Interscience (2006) and the like. To be specific, a protectinggroup having an alkyl group having not less than 10 and not more than300 carbon atoms and/or an alkenyl group having not less than 10 and notmore than 300 carbon atoms in the present invention, as well asphenoxyacetyl group, acetyl group and the like which are protectinggroups of nucleic acid bases can all be removed by a treatment withaqueous ammonia, aqueous ammonia/ethanol solution, or a mixture ofaqueous ammonia and aqueous methylamine solution. In addition, the3′-terminus morpholine ring nitrogen atom-protecting group of themorpholino oligonucleotide can be removed by a treatment with the acidused in step (1′) or an appropriately diluted solution of such acid.

The progress of the reaction in each of the above-mentioned steps can beconfirmed by a method similar to conventional liquid phase organicsynthesis reaction. That is, the reaction can be traced by thin layersilica gel chromatography, high performance liquid chromatography andthe like.

The morpholino oligonucleotide obtained by step (2) or step (3) can alsobe led to a desired morpholino oligonucleotide derivative by furtherapplying an organic synthesis reaction.

The morpholino oligonucleotide produced by the present invention can beused for various applications such as various veterinary pharmaceuticalproducts (RNA, DNA, oligonucleic acid medicine, peptide modifiedmorpholino oligonucleotide etc.) for human or animal, functional food,food for specified health uses, food, chemical product, polymer materialfor living body or industrial use, and the like.

EXAMPLES

The present invention is explained in more detail in the following byreferring to Preparation Examples and Examples, which are not to beconstrued as limiting the scope of the present invention. The reagents,apparatuses and materials used in the present invention are commerciallyavailable unless otherwise specified. In the present specification, whenindicated by abbreviation, unless particularly indicated, eachindication is based on the abbreviation of the IUPAC-IUB Commission onBiochemical Nomenclature or conventional abbreviations in the art.

The abbreviations used in Reference Examples and Examples are asfollows,

-   mo: morpholine nucleoside-   moA: morpholinoadenosine-   moG: morpholinoguanosine-   moC: morpholinocytidine-   moT: morpholinothymidine-   moU; morpholinouridine-   PMO: phosphorodiamidate morpholino oligonucleotide

For example, indication of PMO[A-G-C] means that the left side is the5′-terminus, the right side is the 3′-terminus, and it is aphosphorodiamidate morpholino oligonucleotide in the order ofmorpholinoadenosine, morpholinoguanosine, and morpholinocytidine fromthe 5′-terminus.

-   bz: benzoyl group-   pac: phenoxyacetyl group-   ce: 2-cyanoethyl group

When a nucleic acid base of morpholino nucleoside is protected, theprotecting group is indicated as superscript to the right of theabbreviation (A, G, C, T and U) of the nucleic acid base.

For example, C^(bz) means that the amino group of cytosine is protectedby a benzyl group, and G^(ce/pac) means that the amino group of guanineis protected by a phenoxyacetyl group, and the carbonyl group isprotected by a 2-cyanoethyl group.

-   TOB: 3,4,5-tri(octadecyloxy)benzyloxy group-   suc: succinyl group-   Tr, Trt: trityl group-   CYTFA: cyanopyridine.trifluoroacetate-   DTEA: N,N-diisopropylethylamine-   TFE: 2,2,2-trifluoroethanol-   TFA: trifluoroacetic acid-   DMAP: 4-dimethylaminopyridine-   MeCN: acetonitrile-   HOBt: 1-hydroxybenzotriazole-   UHPLC: ultra high performance liquid chromatography EDC.HCl:    N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride-   MMTr: monomethoxytrityl-   teg: triethylene glycol

Reference Example 1 Synthesis of TOB-suc-moC^(bz)-Tr (Introduction ofsuc-moC^(bz)-Tr Intermediate into TOB Anchor)

3,4,5-tri(Octadecyloxy)benzyl alcohol (4500 mg, 4.92 mmol) was dissolvedin chloroform (45 ml), and suc-moC^(bz)-Tr (3640 mg, 5.41 mmol), EDC.HCl(1141 mg, 5.95 mmol), and DMAP (33 mg, 0.055 mmol) were each added tothe solution in 3 portions every 1 hr in an ice bath. The mixture wasstirred at room temperature overnight, suc-moC^(bz)-Tr (503 mg, 0.74mmol) and EDC.HCl (157 mg, 0.82 mmol) were additionally added thereinand the completion of the reaction was confirmed by HPLC. The solventwas evaporated under reduced pressure, MeCN (10 ml) was added and themixture was evaporated again under reduced pressure. To the residue wasadded MeCN (50 ml), and the precipitate was collected by filtration,completely dissolved in chloroform (30 ml), recrystallized from MeCN (90ml) and dried under reduced pressure to give TOB-suc-moC^(bz)-Tr (7337mg, yield 95%).

TOF-MS+ (m/z)1566.8

Reference Example 2 Detritylation Reaction of TOB-suc-moC^(bz)-Tr.

TOB-suc-moC^(bz)-Tr (500 mg, 0.32 mmol) was dissolved in chloroform (5mL), SOLUTION A* (16 ml) was slowly added dropwise over 30 min in an icebath, and the mixture was stirred at room temperature for 1.5 hr. Aftercompletion of the reaction, a diluted solution of DIEA (137μl)/chloroform (1 mL) was slowly added in an ice bath, MeCN (50 ml) wasadded, and the precipitate was collected by filtration. The obtainedcrystals were washed again with MeCN (10 ml), and dried under reducedpressure to give TOB-suc-moC^(bz) (395 mg, yield 94%).

* composition of SOLUTION A is as described below.

EtOH/TFE/TFA/TEA/chloroform=250 μl/2.5 mL/173 =l/161 μl/22.25 ml

TOF-MS+ (m/z)1324.9

The scheme of Examples 1 and 2 is shown below.

Example 1 Synthesis of Compound 2: Bonding of Succinyl Linker toCompound 1 [mo(Tr)C^(bz)]

Under an argon atmosphere, compound 1 [mo(Tr)C^(bz)] (573 mg, 1.0 mmol)and N,N-dimethylaminopyridine (183 mg, 1.5 mmol) were dissolved in drydichloromethane (7.0 mL), succinic anhydride (150 mg, 1,5 mmol) wasadded, and the mixture was stirred at room temperature for 3 hr. Afterconfirmation of the completion of the reaction by thin layerchromatography, methanol (1.0 mL) was added, and the reaction mixturewas concentrated under reduced pressure. To the obtained concentratedresidue were added ethyl acetate (8.0 mL) and 0.5 mol/l KH₂PO₄ aqueoussolution (8.0 mL) and the mixture was partitioned by extraction.Furthermore, the aqueous layer was extracted with ethyl acetate (8.0mL). The obtained ethyl acetate layers were combined, washed with 0.5mol/l KH₂PO₄ aqueous solution (13.0 mL), water (13.0 mL) and saturatedbrine (13.0 mL), dried over magnesium sulfate, and filtered. Thefiltrate was concentrated to dryness to give compound 2[suc-mo(Tr)C^(bz)] (680 mg, quant).

¹H-NMR (400 MHz, CDCl₃):δ 1.20(1H, t, J=10.4 Hz), 1.49(1H, t, J=11.2Hz), 2.52-2.75(4H, m), 3.14(1H, d, J=11.5 Hz), 3.80(1H, d, J=10.4 Hz),3.92(1H, d, J=11.7 Hz), 4.37-4.59 (2H, m), 6.19(1H, d, J=7.2 Hz),7.15-7.88 (22H, m)

Example 2 Synthesis of Compound 3: Supporting Compound 2[suc-mo(Tr)C^(bz)] on TOB Anchor [3,4,5-tris(octadecyloxy)benzylalcohol]

Under an argon atmosphere, 3,4,5-tris(octadecyloxy)benzyl alcohol (133mg, 0.15 mmol), N,N-dimethylaminopyridine (21 mg, 0.18 mmol), compound 2[suc-mo(Tr)C^(bz)] (123 mg, 0.18 mmol) was dissolved in drydichloromethane (2.4 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (101 mg, 0.53 mmol) was added, and the mixture was stirredat room temperature overnight. After confirmation of the completion ofthe reaction by thin layer chromatography, methanol (6.0 mL) was addedand the obtained mixture was concentrated under reduced pressure. To theconcentrated residue was added methanol (6.0 mL) and the obtained slurrywas stirred for 15 min, and filtered. The obtained solid was dried byheating under reduced pressure to give compound 3[TOB-suc-mo(Tr)C^(bz)](218 mg, 95%).

¹H-NMR (400 MHz, CDCl₃):δ 0.88(9H, t, J=6.3 Hz), 1.22-1.85 (98H, m),2.54-2.68(1H, m), 3.12-3.18(1H, m), 3.58-3.65(1H, m), 3.88-3.98(6H, m),4.07-4.15(2H, m), 4.35-4.43(1H, m), 5.00(2H, s), 6.25-6.29(1H, m),6.53(2H, s), 7.15-7.88(22H, m), 8.50-8.56(1H, m)

m/z 1569.14 [M+H]⁺

The scheme of Examples 3-12 is shown below.

Example 3 Synthesis of Compound 4 [TOB-suc-moC^(bz)] (Method Using CYTFA(Salt of Cyanopyridine and Trifluoroacetic Acid) as Detritylation Agent)

Under an argon atmosphere, dichloromethane solution (1.5 mL) containing2% CYTFA, 1% ethanol, 10% CF₃CH₂OH was added to compound 3[TOB-suc-mo(Tr)C^(bz)] (62.7 mg, 0.04 mmol), and the mixture was stirredat room temperature for 30 min. After confirmation of the completion ofthe reaction by thin layer chromatography, dichloromethane solution (2.3mL) containing 5% N,N-diisopropylethylamine and 25% 2-propanol wasadded, and the mixture was stirred at room temperature for 30 min. Then,methanol (1.5 mL) was added, and the solution was concentrated underreduced pressure. Methanol (1.5 mL) was added again to the concentratedresidue, and the mixture was stirred at room temperature for 15 min. Theobtained slurry was filtered, and the solid was washed with methanol(1.5 mL). The solid was dried by heating in vacuo to give detritylatedcompound 4 [TOB-suc-moC^(bz)] (52.0 mg, 98.0%).

¹H-NMR (400 MHz, CDCl₃):δ 0.88(9H, t, J=6.9 Hz), 1.22-1.85(96H, m),2.45-2.53(1H, m), 2.62-2.74 (5H, m), 2.94-3.00(1H, m), 3.38-3.46(1H, m),3.89-4.00(7H, m), 4.13-4.24(2H, m), 4.98-5.07(2H, m), 5.75-5.82(1H, m),6.54(2H, s), 7.45-7.65(4H, m), 7.83-8.00(3H, m), 8.62(1H, brs)

Example 4 Synthesis of Compound 4 [TOB-suc-moC^(bz)] (Method UsingCyanoacetic Acid as Detritylation Agent)

Under an argon atmosphere, dichloromethane solution (0.75 ml) containing10% cyanoacetic acid and 20% acetonitrile was added to compound 3[TOB-suc-mo(Tr)C^(bz)] (62.7 mg, 0.04 mmol), and the mixture was stirredat room temperature for 30 min. After confirmation of the completion ofthe reaction by thin layer chromatography, dichloromethane solution (4.5mL) containing 5% N,N-diisopropylethylamine, and 25% 2-propanol wasadded, and the mixture was stirred at room temperature for 30 min. Then,methanol (1.5 mL) was added, and the solution was concentrated underreduced pressure. Methanol (1.5 mL) was added again to the concentratedresidue and the mixture was stirred at room temperature for 15 min. Theobtained slurry was filtered, and the solid was washed with methanol(1.5 mL). The solid was dried by heating in vacuo to give detritylatedcompound 4 [TOB-suc-moC^(bz)] (50.0 mg, 94.3%).

Example 5 Synthesis of Compound 5 [TOB-suc-PMO[C^(bz)-T]-Tr] (MethodUsing 3 Equivalents of T Monomer)

Under an argon atmosphere, to compound 4 [TOB-suc-moC^(bz)] (48.9 mg,0.036 mmol) were added a solution (0.4 mL) of ClPONMe₂-mo(Tr)T (61 mg,0.11 mmol) in tetrahydrofuran and 1,3-dimethyl-2-imidazolidinonesolution (0.4 mL) containing 25% N,N-diisopropylethylamine, and themixture was stirred at room temperature for 1.5 hr. After confirmationof the completion of the reaction by thin layer chromatography, methanol(1.5 mL) was added to the reaction mixture, and the solution wasconcentrated under reduced pressure. To the concentrated residue wasadded methanol (1.5 mL), and the mixture was stirred at room temperaturefor 15 min. The obtained slurry was filtered, and the solid was washedwith methanol (1.5 mL). The solid was dried by heating in vacuo to givecompound 5 [TOB-suc-PMO[C^(bz)-T]-Tr] (66.0 mg, 99.0%).

Example 6 Synthesis of Compound 5 [TOB-suc-PMO[C^(bz)-T]-Tr] (MethodUsing T Monomer Decreased to 1.1 Equivalents)

Under an argon atmosphere, to compound 4 [TOB-suc-moC^(bz)] (54.2 mg,0.04 mmol) were added a solution (0.4 mL) of ClPONMe₂-mo(Tr)T (27 mg,0.044 mmol) in tetrahydrofuran and 1,3-dimethyl-2-imidazolidinonesolution (0.4 mL) containing 25% N,N-diisopropylethylamine, and themixture was stirred at room temperature for 1.5 hr. After confirmationof the completion of the reaction by thin layer chromatography, methanol(1.5 mL) was added to the reaction mixture, and the solution wasconcentrated under reduced pressure. To the concentrated residue wasadded methanol (1.5 mL) and the mixture was stirred at room temperaturefor 15 min. The obtained slurry was filtered, and the solid was washedwith methanol (1.5 mL). The solid was dried by heating in vacuo to givecompound 5 [TOB-suc-PMO[C^(bz)-T]-Tr] (70.8 mg, 93.2%).

Example 7 Synthesis of Compound 5 [TOB-suc-PMO[C^(bz)-T]-Tr] (One-PotMethod from Compound 3)

Under an argon atmosphere, to compound 3 [TOB-suc-mo(Tr)C^(bz)] (62.7mg, 0.04 mmol) was added dichloromethane solution (1.8 mL) containing 2%CYTFA, 1% ethanol, 10% CF₃CH₂OH, and the mixture was stirred at roomtemperature for 30 min. After confirmation of the completion of thereaction by thin layer chromatography, 5% N,N-diisopropylethylamine, anddichloromethane solution (2.7 mL) containing 25% 2-propanol were added,and the mixture was stirred at room temperature for 30 min. Then, to thereaction mixture were added a solution (0.4 mL) of ClPONMe₂-mo(Tr)T(73.1 mg, 0.12 mmol) in tetrahydrofuran and1,3-dimethyl-2-imidazolidinone solution (0.5 mL) containing 25%N,N-diisopropylethylamine, and the mixture was stirred at roomtemperature for 30 min, and further at 38° C. for 1 hr. Methanol (1.5mL) was added to the reaction mixture, and the solution was concentratedunder reduced pressure. To the concentrated residue was added methanol(1.5 mL), and the mixture was stirred at room temperature for 15 min.The obtained slurry was filtered, and the solid was washed with methanol(1.5 mL). The solid was dried by heating in vacuo to give compound 5[TOB-suc-PMO[C^(bz)-T]-Tr] (75.0 mg, 98.8%).

Example 8 Synthesis of Compound 6 [TOB-suc-PMO[C^(bz)-T]] (Method UsingCYTFA as Detritylation Agent)

Under an argon atmosphere, to compound 5 [TOB-suc-PMO[C^(bz)-T]-Tr](37.4 mg, 0.02 mmol) was added dichloromethane solution (0.75 ml)containing 2% CYTFA, 1% ethanol, 10% CF₃CH₂OH, and the mixture wasstirred at room temperature for 30 min. After confirmation of thecompletion of the reaction by thin layer chromatography, dichloromethanesolution (1.0 mL) containing 5% N,N-diisopropylethylamine, 25%2-propanol was added, and the mixture was stirred at room temperaturefor 30 min. Then, methanol (1.5 mL) was added, and the solution wasconcentrated under reduced pressure. Methanol (1.5 mL) was added againto the concentrated residue and the mixture was stirred at roomtemperature for 15 min. The obtained slurry was filtered, and the solidwas washed with methanol (1.5 mL). The solid was dried by heating invacuo to give detritylated compound 6 [TOB-suc-PMO[C^(bz)-T]] (30.0 mg,92.1%).

Example 9 Synthesis of Compound 6 [TOB-suc-PMO[C^(bz)-T]] (Method UsingCyanoacetic Acid as Detritylation Agent)

Under an argon atmosphere, dichloromethane solution (0.38 ml) containing10% cyanoacetic acid and 20% acetonitrile was added to compound 5[TOB-suc-PMO[C^(bz)-T]-Tr] (37.4 mg, 0.02 mmol), and the mixture wasstirred at room temperature for 30 min. After confirmation of thecompletion of the reaction by thin layer chromatography, dichloromethanesolution (2.3 mL) containing 5% N,N-diisopropylethylamine and 25%2-propanol was added, and the mixture was stirred at room temperaturefor 30 min. Then, methanol (1.5 mL) was added, and the solution wasconcentrated under reduced pressure. Methanol (1.5 mL) was added againto the concentrated residue, and the mixture was stirred at roomtemperature for 15 min. The obtained slurry was filtered, and the solidwas washed with methanol (1.5 mL). The solid was dried by heating invacuo to give detritylated compound 6 [TOB-suc-PMO[C^(bz)-T]] (31.0 mg,95.2%).

Example 10 Synthesis of Compound 7 [TOB-suc-PMO[C^(bz)-T-C^(bz)]-Tr](Method Using 3 Equivalents of C Monomer)

Under an argon atmosphere, to compound 6 [TOB-suc-PMO[C^(bz)-T]] (25.8mg, 0.016 mmol) were added a solution (0.2 mL) of ClPONMe₂-mo(Tr)C^(bz)(32.7 mg, 0.05 mmol) in tetrahydrofuran and1,3-dimethyl-2-imidazolidinone solution (0.2 mL) containing 25%N,N-diisopropylethylamine, and the mixture was stirred at roomtemperature for 1.5 hr. After confirmation of the completion of thereaction by thin layer chromatography, methanol (1.5 mL) was added tothe reaction mixture, and the solution was concentrated under reducedpressure. To the concentrated residue was added methanol (1.5 mL), andthe mixture was stirred at room temperature for 15 min. The obtainedslurry was filtered, and the solid was washed with methanol (1.5 mL).The solid was dried by heating in vacuo to give compound 7[TOB-suc-PMO[C^(bz)-T-C^(bz)]-Tr] (32.9 mg, 90.9%).

Example 11 Synthesis of Compound 7 [TOB-suc-PMO[C^(bz)-T-C^(bz)]-Tr](Method Using C Monomer Decreased to 1.5 Equivalents)

Under an argon atmosphere, to compound 6 [TOB-suc-PMO[C^(bz)-T]] (99.4mg, 0.06 mmol) were added a solution (0.6 mL) of ClPONMe₂-mo(Tr)C^(bz)(62.8 mg, 0.09 mmol) in tetrahydrofuran and1,3-dimethyl-2-imidazolidinone solution (0.75 mL) containing 25%N,N-diisopropylethylamine, and the mixture: was stirred at roomtemperature for 5 hr. After confirmation of the completion of thereaction by thin layer chromatography, methanol (2.7 mL) was added tothe reaction mixture, and the solution was concentrated under reducedpressure. To the concentrated residue was added methanol (2.7 mL) andthe mixture was stirred at room temperature for 15 min. The obtainedslurry was filtered, and the solid was washed with methanol (2.7 mL) anddried by heating in vacuo to give compound 7[TOB-suc-PMO[C^(bz)-T-C^(bz)]-Tr] (131 mg, 94.4%).

Example 12 Synthesis of Compound 7 [TOB-suc-PMO[C^(bz)-T-C^(bz)]-Tr](One-Pot Method from Compound 5)

Under an argon atmosphere, to compound 5 [TOB-suc-PMO[C^(bz)-T]-Tr](75.9 mg, 0.04 mmol) was added dichloromethane solution (1.5 mL)containing 2% CYTFA, 1% ethanol, 10% CF₃CH₂OH, and the mixture wasstirred at room temperature for 30 min. After confirmation of thecompletion of the reaction by thin layer chromatography, dichloromethanesolution (2.3 mL) containing 5% N,N-diisopropylethylamine and 25%2-propanol was added, and the mixture was stirred at room temperaturefor 30 min. Then, to the reaction mixture were added a solution (0.4 mL)of ClPONMe₂-mo(Tr)C^(bz) (83.8 mg, 0.12 mmol) in tetrahydrofuran and1,3-dimethyl-2-imidazolidinone solution (0.5 mL) containing 25%N,N-diisopropylethylamine, and the mixture was stirred at roomtemperature for 30 min, and further stirred at 38° C. for 45 min.Methanol (1.5 mL) was added to the reaction mixture, and the solutionwas concentrated under reduced pressure. To the concentrated residue wasadded methanol (1.5 mL), and the mixture was stirred at room temperaturefor 15 min. The obtained slurry was filtered, and the solid was washedwith methanol (1.5 mL) and dried by heating in vacuo to give compound 7[TOB-suc-PMO[C^(bz)-T-C^(bz)]-Tr] (90.8 mg, 97.9%).

The scheme of Example 13 is shown below.

Example 13 Synthesis of Compound 8 [PMO[CTC]]

To compound 7 [TOB-suc-PMO[C^(bz)-T-C^(bz)]-Tr] (162 mg, 0.07 mmol) wasadded a mixture of 28% aqueous ammonia (3.5 mL) and ethanol (14.0 mL),and the mixture was sealed in an autoclave and Stirred with heating at55° C. overnight. The mixture was cooled to room temperature, and thecompletion of the reaction was confirmed by thin layer chromatography.The content was transferred into methanol while washing with methanoland dichloromethane. The mixture was concentrated under reducedpressure, methanol (14.0 mL) was added to the obtained concentratedresidue, and the mixture was slurry washed for 30 min. The solid wasfiltered off and the filtrate was concentrated. The obtained solid wasdissolved in 0.2 mol/l KH₂PO₄ aqueous solution (pH 2.0), and the mixturewas stirred for 15 min. The disappearance of the starting material wasconfirmed by thin layer chromatography, and the mixture was adjusted topH13-pH14 with 2.0 mol/l aqueous sodium hydroxide solution, and filteredthrough a membrane filter. As a result of HPLC analysis, the filtratewas identical to the object analysis reference standard.

Reversed-phase HPLC analysis (Column=InertSustain C18, 5 μm, 4.6×150 mm;Solvent A=50 mM TEAA (pH 7.0), Solvent B═CH₃CN; Gradient=0% to 40% B (20min); Flow Rate=0.75 ml/min; Temp=60° C.): RT 11.6 min, 11.7 min, 11.8min, 12.0 min (4 isomer mixture)

The scheme of Examples 14-16 is shown below.

Example 14 Synthesis of Compound 9[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(pac)-G^(pac)-T-T-C^(bz)-T-G^(pac)]-Tr](One Time Isolation/1 Cycle Method)

Deprotection of trityl group and coupling of the corresponding monomerwere sequentially performed in one pot from compound 3[TOB-suc-mo(Tr)C^(bz)] (78.4 mg, 0.05 mmol) according to the methodsdescribed in Examples 7 and 12. The weight and weight yield of theresultant product isolated in each stage are shown in the followingTable.

TABLE 1 chain amount weight length obtained yield  2mer  96 mg 97%  3mer104 mg 95%  4mer 120 mg 100%   5mer 134 mg 104%   6mer 138 mg 92%  7mer151 mg 104%   8mer 139 mg 89%  9mer 110 mg 84% 10mer 101 mg 97% 11mer 70 mg 67% 12mer  74 mg 94%

Example 15 Synthesis of Compound 9[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(pac)-G_(pac)-T-T-C^(bz)-T-G^(pac)-]-Tr](2 Time Isolation/1 Cycle, Method Using Cyanoacetic Acid asDetritylation Agent)

Deprotection of trityl group from compound 3 [TOB-suc-mo(Tr)C^(bz)](94-1 mg, 0.06 mmol) was performed according to the methods described inExamples 4 and 9 and coupling of the corresponding monomer wassequentially performed according to the methods described in Examples 5and 10. The weight and weight yield of the resultant product isolated ineach stage are shown in the following Table.

TABLE 2 chain amount weight length obtained yield 2mer 112 mg 94% 3mer169 mg 94% 4mer 183 mg 94% 5mer 206 mg 98% 6mer 216 mg 93% 7mer 231 mg95% 8mer 236 mg 96% 9mer 245 mg 97% 10mer  232 mg 88% 11mer  222 mg 93%12mer  218 mg 90%

Example 16 Synthesis of Compound 10 [PMO[CCTCCGGTTCTG]-Tr]

To compound 9[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(pac)-G^(pac)-T-T-C^(bz)-T-G^(pac)]-Tr](6.0 mg, 1.0 μmol) synthesized in Example 15 was added 28% aqueousamraonia/ethanol=1/3(v/v) (0.2 mL), and the mixture was sealed in anautoclave, and stirred with heating at 55° C. overnight. The mixture wasallowed to cool to room temperature, and filtered while washing withmethanol to remove the solid. The filtrate was concentrated to give acrude product (3.3 mg, 80%) of compound 10 [PMO[CCTCCGGTTCTG]-Tr].

reversed-phase HPLC analysis (Column=InertSustain C18, 5 μm, 4.6×150 mm;Solvent A=50 mM TEAA (pH 7.0), Solvent B=CH₃CN; Gradient=10% to 70% B(20 min); Flow Rate=0.75 ml/min; Temp=60° C.): RT 13.1 min

The scheme of Examples 17 and 18 is shown below.

Example 17 Synthesis of Compound 11[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(pac)-G^(pac)-T-T-C^(bz)-T-G^(pac)-A^(bz)-A^(bz)-G^(pac)-G^(pac)-T-G^(pac)-T-T-C^(bz)]-Tr]

Deprotection of trityl group from compound 3 [TOB-suc-mo(Tr)C^(bz)](78.4 mg, 0.05 mmol) was performed according to the methods described inExamples 3 and 8, and coupling of the corresponding monomer wassequentially performed according to the methods described in Examples 5and 10. The weight and weight yield of the resultant product isolated ineach stage are shown in the following Table.

TABLE 3 chain length amount obtained weight yield  2mer 92 mg 92%  3mer89 mg 86%  4mer 98 mg 95%  5mer 103 mg  91%  6mer 111 mg  95%  7mer 123mg  98%  8mer 112 mg  85%  9mer 103 mg  87% 10mer 95 mg 86% 11mer 93 mg94% 12mer 92 mg 92% 13mer 87 mg 90% 14mer 84 mg 90% 15mer 82 mg 92%16mer 79 mg 91% 17mer 71 mg 86% 18mer 65 mg 86% 19mer 49 mg 74% 20mer 37mg 75% 21mer 25 mg 66%

Example 18 Synthesis of Compound 12 [PMO[CCTCCGGTTCTGAAGGTGTTC]-Tr]

Compound 11[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(pac)-G^(pac)-T-T-C^(bz)T-G^(pac)-A^(bz)-A^(bz)-G^(pac)-G^(pac)-T-G^(pac)-T-T-C^(bz)]-Tr](6 mg, 0.61 mmol) was dissolved in 28% aqueous ammonia/ethanol=1/3 (v,v)(200 μl), and the mixture was sealed in an autoclave and stirred at 55°C. overnight. The mixture was allowed to cool to room temperature, andfiltered while washing with methanol to remove the solid. The filtratewas concentrated to give a crude product (3.1 mg, 71%) of compound 12[PMO[CCTCCGGTTCTGAAGGTGTTC]-Tr].

Reversed-phase HPLC analysis (Column=Inert Sustain C18, 5 μm, 4.6×150mm; Solvent A=50 mM TEAA (pH 7.0), Solvent B═CH₃CN; Gradient=10% to 70%B (20 min); Flow Rate=0.75 ml/min; Temp=60° C.): RT 12.4 min

Anion exchange HPLC analysis (Column=DNAPacPA-100, 4.0×250 mm; SolventA=10 mM NaOH aqueous solution, B=1M NaCl, 10 mM NaOH aqueous solution;Gradient=20% to 100% B (30 min);

Flow Rate=1.0 mL/min; Temp=35° C.): RT 10.8 min

m/z 7167.50 [M+H]⁻

The scheme of Examples 19 and 20 is shown below.

Example 19 Synthesis of Compound 13[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T]-Tr]

Deprotection of trityl group from compound 3 [TOB-suc-mo(Tr)C^(bz)] (502mg, 0.32 mmol) was performed according to the methods described inExamples 3 and 8, and coupling of the corresponding monomer wassequentially performed according to the methods described in Examples 5and 10. As a monomer corresponding to guano sine,ClPONMe₂-mo(Tr)G^(ce/pac) having a cyanoethyl group at the O6-positionas a protecting group was used. The weight and weight yield of theresultant product isolated in each stage are shown in the followingTable.

TABLE 4 chain length amount obtained weight yield 2mer 536 mg 87% 3mer576 mg 94% 4mer 632 mg 96% 5mer 694 mg 97% 6mer 732 mg 92% 7mer 682 mg92% 8mer 572 mg 89%

Example 20 Synthesis of Compound 14 [PMO[CCTCCGGT]-Tr]

Compound 13[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T]-Tr](19.3 mg, 4.2 μmol) synthesized in Example 19 was suspended in 28%aqueous amisonia/ethanol=1/3 (v/v) (2.0 mL), and the suspension wassealed in an autoclave and stirred with heating overnight at 55° C.After allowing to cool to room temperature, the liquid was transferredusing dichloromethane (1.5 mL) and methanol (1.5 mL) into aneggplant-shaped flask, and concentrated under reduced pressure. To theconcentrated residue was added methanol (2.0 mL) and the precipitatedsolid was filtered through a membrane filter. The filtrate wasconcentrated to dryness to quantitatively give compound 14[PMO[CCTCCGGT]-Tr].

m/z, 929.2 [M+3H]³⁺, 1393.3 [M+2H]²⁺

The scheme of Examples 21 and 22 is shown below.

Example 21 Synthesis of compound 15[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)-T-G^(ce/pac)-A^(bz)]-Tr]

Using compound 13[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)G^(ce/pac)-G^(ce/pac)-T]-Tr](487 mg, 0.11 mmol) synthesized in Example 19 and according to themethod described in Example 12, deprotection of trityl group andcoupling with the corresponding monomer were performed. As a monomercorresponding to guanosine, ClPONMe₂-mo(Tr)G^(ce/pac) having acyanoethyl group at the O6-position as a protecting group was used. Theweight and weight yield of the resultant product isolated in each stageare shown in the following Table.

TABLE 5 chain length amount obtained weight yield  9mer 429 mg 86% 10mer400 mg 93% 11mer 308 mg 86% 12mer 267 mg 86% 13mer 208 mg 92%

Example 22 Synthesis of Compound 16 [PMO[CCTCCGGTTCTGA]-Tr]

Compound 15[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)-T-G^(ce/pac)-A^(bz)]-Tr](8.6 mg, 1.3 μmol) synthesized in Example 21 was suspended in 28%aqueous ammonia/ethanol=1/3 (v/v) (1.0 mL), and the suspension wassealed in an autoclave and stirred with heating overnight at 55° C.After allowing to cool to room temperature, the liquid was transferredusing dichloromethane (1.0 mL) and methanol (1.0 mL) into aneggplant-shaped flask, and concentrated under reduced pressure. To theconcentrated residue was added methanol (1.0 mL) and the precipitatedsolid was filtered off through a membrane filter. The filtrate wasconcentrated to dryness to quantitatively give compound 16[PMO[CCTCCGG]-Tr].

m/z 1114.8 [M+4H]⁴⁺, 1486.0 [M+3H]³⁺

The scheme of Examples 23 and 24 is shown below.

Example 23 Synthesis of Compound 17[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)]-Tr]

Under an argon atmosphere, to compound 3 [TOB-suc-mo(Tr)C^(bz)] (300 mg,0.19 mmol) was added dichloromethane solution (8.0 ml) containing 2%CYTFA, 1% ethanol, 10% CF₃CH₂OH, and the mixture was stirred at roomtemperature for 90 min. After confirmation of the completion of thereaction by thin layer chromatography, dichloromethane solution (12.0mL) containing 5% N,N-diisopropylethylamine and 25% 2-propanol wasadded, and the mixture was stirred at room temperature for 30 min. Thissolution was concentrated under reduced pressure. Acetonitrile (8.0 mL)was added and the mixture was stirred at room temperature for 15 min.The obtained slurry was filtered, and the solid was washed withacetonitrile (8.0 mL) and dried by heating in vacuo to give detritylatedcompound 4 [TOB-suc-moC^(bz)] (254 mg, 100%).

Under an argon atmosphere, to compound 4 [TOB-suc-moC^(bz)] (242 mg,0.18 mmol) were added a solution of ClPONMe₂-mo(Tr)C^(bz) (382 mg, 0.55mmol) in tetrahydrofuran (1.8 mL) and 1,3-dimethyl-2-imidazolidinonesolution (2.0 mL) containing 25% N,N-diisopropylethylamine, and themixture was stirred at room temperature for 30 min. After confirmationof the completion of the reaction by thin layer chromatography,morpholine (48 μL, 0.55 mmol) was added to the reaction mixture, and themixture was stirred for 10 min. Then, acetonitrile (7.3 mL) was added tothe solution and the mixture was concentrated under reduced pressure. Tothe concentrated residue was added acetonitrile (7.3 mL) again, and theobtained slurry was stirred for 15 min. The slurry was filtered, and theobtained solid was washed with acetonitrile (7.3 mL) and dried byheating under reduced pressure to give TOB-suc-PMO[Cbz-Cbz]-Tr (355 mg,98%).

Hereafter, detritylation and coupling with the corresponding monomerwere sequentially performed to give the title compound 17[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)]-Tr].The weight and weight yield of the resultant product isolated in eachstage are shown in the following Table.

TABLE 6 chain length amount obtained weight yield 2mer 355 mg 98% 3mer399 mg 110%  4mer 358 mg 87% 5mer 372 mg 100%  6mer 386 mg 98% 7mer 306mg 77%

Example 24 Synthesis of Compound 18 [PMO[CCTCCGG]-Tr]

The compound 17[TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)]-Tr] (4mg, 0.94 μmol) synthesized in Example 23 was subjected to deprotectionaccording to the method described in Example 22 and detachment from thecarrier to give compound 18 [PMO[CCTCCGG]-Tr].

m/z 819.3 [M+3H]³⁺, 1228.4 [M+2H]²⁺

Example 25 [TOB-suc-PMO[C^(bz)-C^(bz)]] (One-Pot Elongation)

TOB-suc-moC^(bz) (395 mg, 0.30 mmol) was dissolved in dichloromethane (4mL), DIEA (1.8 eq, 0.54 mmoL, 50 μl), and ClPONMe₂-mo(Tr)C^(bz) (1.5 eq,0.45 mmoL, 312 mg) were added in 3 portions at 30 min intervals in anice bath, and the mixture was stirred at room temperature overnight.Then, DIEA (0.1 eq, 0.03 mmoL, 5 μl), ClPONMe₂-mo(Tr)C^(bz) (0.1 eq,0.03 mmoL, 21 mg) were added 3 times each, and the mixture was furtherstirred overnight. After completion of the reaction, morpholine (1.28eq, 0.37 mmoL, 34 μl) was added, and the mixture was stirred for 1.5 hr.The disappearance of remaining ClPONMe₂-mo(Tr)C^(bz) was confirmed,SOLUTION A (16.8 mL) of Reference Example 2 was slowly added dropwiseover 10 min, and the mixture was stirred for 2 hr at room temperature.After completion of the detritylation reaction, a diluted solution ofDIEA (142 μL, 1.0 eq vs TFA)/dichloromethane (1 mL) was slowly added inan ice bath, and MeCN (50 ml) was added to allow for precipitation. Theprecipitate in the suspension was filtered, and the filtrate was washedwith MeCN to give [TOB-suc-PMO[C^(bz)-C^(bz)]] (wet. 526 mg;).

TOF-MS+ (m/z) 1743.8

Example 26 Synthesis of [TOB-suc-PMO[C^(bz)-C^(bz)-T]] (Condensation 3and One-Pot Elongation)

[TOB-suc-PMO[C^(bz)-C^(bz)]] as wet crystals (wet. 526 mg) was dissolvedin dichloromethane (10 ml) in an ice bath, DIEA (0.54 mmol, 50 μl), andClPONMe₂-mo(Tr)T (0.45 mmol, 272 mg) was added in 3 portions at 30 minintervals. The mixture was stirred at room temperature overnight. Then,DIEA (0.03 mmol, 3 μl), and ClPONMe₂-mo(Tr)T (0.03 mmol, 17 mg) wereadded twice, and the mixture was stirred at room temperature for 4 hr.After completion of the reaction, morpholine (0.5 eq, 0.14 mmoL, 21 μl)was added at ambient temperature, and the mixture was stirred for 3 hr.SOLUTION A (30 ml) of Reference Example 2 was slowly added dropwise over15 min in an ice bath and, after the completion of the dropwiseaddition, the mixture was stirred at room temperature for 4 hr. Aftercompletion of the reaction, a diluted solution of DIEA (77μl)/dichloromethane (1 ml) was slowly added in an ice bath and MeCN (50ml) was added to give a white slurry. 25 ml of this liquid wasevaporated under reduced pressure, MeCN (20 ml) was added, and theslurry was stirred for 15 min and filtered. The obtained crystals werewash again with MeCN (30 ml) and dried under reduced pressure to give[TOB-suc-PMO[C^(bz)-C^(bz)-T]] (yield 95%) (vs TOB-suc-moC^(bz)).

TOF-MS+ (m/z) 2073.4

Comparative Example 1 Evaluation of Influence of Quenching of ExcessMonomer by Morpholine on Quality

The 2 mer to 5 mer intermediates obtained on the way of synthesisdescribed in Example 14 were subjected to deprotection by the methoddescribed in Example 14 and detachment from the carrier, and impurityanalysis was performed by mass spectrometry (LC-MS). Similarly, the 2mer to 7 mer intermediates obtained on the way of synthesis described inExample 23 were also subjected to deprotection and detachment from thecarrier, and impurity analysis was performed by mass spectrometry(LC-MS). The respective analysis results were compared. It was foundthat the method described in Example 23 wherein excess monomer wasquenched by morpholine was free of byproduction of impurity in excesselongation such as n+1 mer and the like.

TABLE 7 without morpholine quenching with morpholine quenching chainobject chain object length product (n + x) length product (n + x) 2merCC not 2mer CC not detected detected 3mer CCT CCT + C was 3mer CCT notobserved detected 4mer CCTC CCTC + C, 4mer CCTC trace CCTC + T wereamount of observed CCTC + C, CCTC + T were observed 5mer CCTCC CCTCC +C, 5mer CCTCC not CCTCC + T detected were observed 6mer CCTCCG notdetected 7mer CCTCCGG not detected * Indication of terminal trityl groupis omitted.

Example 27 Synthesis of DPM-suc-mo(Tr)C^(bz)

α,α-Di-4,4′-docosyloxyphenylmethylamine (1.0 g, 1.20 mmol) was dissolvedin chloroform (20 mL), HOBt (162.3 mg, 1.20 mmol), suc-mo(Tr)C^(bz)(969.8 mg, 1.44 mmol) and EDC.HCl (152.0 mg, 0.79 mmol) were added andthe mixture was stirred at room temperature for 20 min. Then, to thereaction mixture was added again EDC.HCl (152.0 mg, 0.79 mmol), and themixture was stirred at room temperature for 24 hr. After confirmation ofthe completion of the reaction by UHPLC, the solvent was evaporatedunder reduced pressure. To the obtained residue was added acetonitrile(10 mL), and the precipitates were collected by filtration. The obtainedcrystals were washed again with acetonitrile (10 mL) and dried underreduced pressure to give DPM-suc-mo(Tr)C^(bz) (1.70 g, yield 9 6%).

TOF-MS+ (m/z) 1486.9

The scheme of Examples 28 and 29 is shown below.

Example 28 Synthesis of DPM-suc-moC^(bz)

DPM-suc-mo(Tr)C^(bz) (1.69 g, 1.13 mmol) was dissolved in chloroform(16.9 mL), and ice-cooled. 2,2,2-Trifluoroethanol (4.3 mL) and ethanol(0.52 g, 11.3 mmol) were added, a solution of trifluoroacetic acid (1.03g, 9.07 mmol) and triethylamine (0.57 g, 5.67 mmol) in chloroform (7.0mL) was added dropwise, and the mixture was stirred at 15° C. for 1 hr.After confirmation of the completion of the reaction by UHPLC, thereaction mixture was ice-cooled and a solution ofN,N-diisopropylethylamine (0.59 g, 4.54 mmol) in chloroform (6 mL) wasadded dropwise. To the reaction mixture was added acetonitrile (16.9 mL)and the solvent was evaporated under reduced pressure. To the obtainedresidue was added acetonitrile (16.9 mL), and the mixture was stirred at0° C. for 30 min, and the precipitates were collected by filtration. Theobtained crystals were washed again with acetonitrile (16.9 mL) to giveDPM-suc-moC^(bz). The crystals were directly used without drying as astarting material of the next reaction.

TGF-MS+ (m/z) 1244.9

Example 29 Synthesis of DPM-suc-PMO[C^(bz)-C^(bz)]-Tr

DPM-suc-moC^(bz) wet crystals (3.33 g, corresponding to 1.13 mmol) weredissolved in chloroform (9.8 mL), and cyclohexane (4.2 mL) was added.N,N-diisopropylethylamine (0.26 g, 2.03 mmol) and ClPONMe-mo(Tr)C^(bz)(1.18 g, 1.69 mmol) were added, and the mixture was stirred at 40° C.for 21 hr with heating. After confirmation of the completion of thereaction by UHPLC, the reaction mixture was cooled to room temperature,morpholine (0.20 g, 2.25 mmol) was added and the mixture was stirred for1 hr. To the reaction mixture was added acetonitrile (14 mL), and thesolvent was evaporated under reduced pressure. The obtained slurry wasstirred at 0° C. and the precipitates were collected by filtration. Theobtained crystals were washed again with acetonitrile (14 mL), and driedunder reduced pressure to give DPM-suc-PMO[C^(bz)-C^(bz)]-Tr (1.96 g,yield 91%).

TOF-MS+ (m/z) 1906.3

Example 30 Synthesis of TOB-suc-PMO[C^(bz)-C^(bz)]-MMTr

TOB-suc-mo(Tr)C^(bz) (1.0 g, 0.64 mmol) was dissolved in chloroform (10mL), and ice-cooled. 2,2,2-trifluoroethanol (2.8 mL) and ethanol (0.29g, 6.4 mmol) were added, a solution of trifluoroacetic acid (0.29 g,2.55 mmol) and triethylamine (0.14 g, 1.27 mmol) in chloroform (4.5 mL)was added dropwise, and the mixture was stirred at room temperature for2 hr. After confirmation of the completion of the reaction by UHPLC, thereaction mixture was ice-cooled, and a solution ofN,N-diisopropylethylamine (0.33 g, 2.55 mmol) in chloroform (3 mL) wasadded dropwise. To the reaction mixture was added acetonitrile (30 mL),and the solvent was evaporated under reduced pressure. To the obtainedresidue was added acetonitrile (20 mL), and the mixture was stirred at0° C. for 30 min, and the precipitates were collected by filtration. Theobtained crystals were washed again with acetonitrile (20 mL) to give:TOB-suc-moC^(bz). The crystals were directly used without drying as astarting material of the next reaction.

TOS-suc-moC^(bz) wet crystals (1.39 g, corresponding to 0.64 mmol) weredissolved in chloroform (10 mL), N,N-diisopropylethylamine (0.15 g, 1.15mmol) and ClPONMe₂-mo(MMTr)C^(bz) (695 mg, 0.96 mmol) were added, andthe mixture was stirred at room temperature for 17 hr. Afterconfirmation of the completion of the reaction by UHPLC, to the obtainedsolution was added acetonitrile (20 mL), and the precipitates werecollected by filtration. The obtained crystals were washed again withacetonitrile (20 mL) and dried under reduced pressure to giveTOB-suc-PMO[C^(bz)-C^(bz)]-MMTr (1.19 g, yield 92%).

TOF-MS+ (m/z) 2017.3

Example 31 Synthesis of TOB-suc-PMO[C^(bz)-T] (Elongation-2)

TOB-suc-moC^(bz) (4.3 g, 3.23 mmol) was dissolved in chloroform (33 mL),N,N-diisopropylethylamine (0.73 g, 5.81 mmol) and ClPONMe₂-mo(Tr)T (2.95g, 4.85 mmol) were added, and the mixture was stirred at roomtemperature for 19 hr. After confirmation of the completion of thereaction by UHPLC, the reaction mixture was cooled to room temperature,morpholine (0.23 g, 3.23 mmol) was added and the mixture was stirred atroom temperature for 1 hr. The reaction mixture was ice-cooled,2,2,2-trifluoroethanol (13.1 mL) and ethanol (1.49 g, 32.3 mmol) wereadded, a solution of trifluoroacetic acid (2.95 g, 25.8 mmol) andtriethylamine (1.21 g, 12.0 mmol) in chloroform (9.9 mL) was addeddropwise, and the mixture was stirred at 15° C. for 1.5 hr. Afterconfirmation of the completion of the reaction by UHPLC, the reactionmixture was ice-cooled, and a solution of N,N-diisopropylethylamine(3.26 g, 25.8 mmol) in chloroform (4.5 mL) was added dropwise. To thereaction mixture was added acetonitrile (86 mL), and the solvent wasevaporated under reduced pressure. The obtained slurry was stirred at 0°C. for 30 min, and the precipitates were collected by filtration. Theobtained crystals were; washed again with acetonitrile (43 mL) to giveTOB-suc-PMO[C^(bz)-T]. The crystals were directly used without drying asa starting material of the next reaction.

Example 32 Synthesis of TOB-suc-PMO[C^(bz)-T-C^(bz)] (Elongation-3)

TOB-suc-PMO[C^(bz)-T] wet crystals (5.7 g, corresponding to 3.20 mmol)were dissolved in chloroform (43 mL), N,N-diisopropylethylamine (0.73 g,25.6 mmol) and ClPONMe₂-mo(Tr)C^(bz) (3.35 g, 4.8 mmol) were added, andthe mixture was stirred at room temperature for 16 hr. Afterconfirmation of the completion of the reaction by UHPLC, the reactionmixture was cooled to room temperature, morpholine (0.28 g, 3.20 mmol)was added and the mixture was stirred at room temperature for 1 hr. Thereaction mixture was ice-cooled, 2,2,2-trifluoroethanol (13.0 mL) andethanol (1.48 g, 32.0 mmol) were added, a solution of trifluoroaceticacid (2.92 g, 25.6 mmol) and triethylamine (1.20 g, 11.8 mmol) inchloroform (9.8 mL) was added dropwise, and the mixture was stirred at15° C. for 1.5 hr. After confirmation of the completion of the reactionby UHPLC, the reaction mixture was ice-cooled, and a solution ofN,N-diisopropylethylamine (3.23 g, 25.6 mmol) in chloroform (4.5 mL) wasadded dropwise. To the reaction mixture was added acetonitrile (106 mL),and the solvent was evaporated under reduced pressure. The obtainedslurry was stirred at 0° C. for 15 min, and the precipitates werecollected by filtration. The obtained crystals were washed again withacetonitrile (53 mL) to give TOB-suc-PMO[C^(bz)-T-C^(bz)]. The crystalswere directly used without drying as a starting material of the nextreaction.

Example 33 Synthesis of TOB-suc-PMO[C^(bz)-T] (Lithium Chloride AdditionSystem, Elongation-2)

TOB-suc-moC^(bz) wet crystals (4.9 g, corresponding to 3.08 mmol) weredissolved in chloroform (34.9 mL), lithium chloride (143 mg, 3.38 mmol),N,N-diisopropylethylamine (0.72 g, 5.54 mmol) and ClPONMe₂-mo(Tr)T (2.81g, 4.61 mmol) were added, and the mixture was stirred at 40° C. for 16hr. After confirmation of the completion of the reaction by UHPLC, thereaction mixture was cooled to room temperature, morpholine (0.32 g,3.69 mmol) was added and the mixture was stirred at room temperature for1 hr. The reaction mixture was ice-cooled, 2,2,2-trifluoroethanol (12.5mL) and ethanol (1.41 g, 30.8 mmol) were added, and then, a solution oftrifluoroacetic acid (1.75 g, 15.4 mmol) and triethylamine (0.49 g, 4.86mmol) in chloroform (5.9 mL) was added dropwise, and the mixture wasstirred at room temperature for 1 hr. After confirmation of thecompletion of the reaction by UHPLC, the reaction mixture wasice-cooled, and a solution of N,N-diisopropylethylamine (2.0 g, 15.4mmol) in chloroform (2.7 mL) was added dropwise. To the reaction mixturewas added acetonitrile (82 mL), and the solvent was evaporated underreduced pressure. The obtained slurry was stirred at 0° C. for 30 min,and the precipitates were collected by filtration. The obtained crystalswere washed again with acetonitrile (41 mL) to giveTOB-suc-PMO[C^(bz)-T]. The crystals were directly used without drying asa starting material of the next reaction.

Example 34 Synthesis of TOB-suc-PMO[C^(bz)-T-C^(bz)] (Lithium ChlorideAddition System, Elongation-3)

TOB-suc-PMO[C^(bz)-T] crystals (5.5 g, corresponding to 3.05 mmol) weredissolved in chloroform (44.6 mL), lithium chloride (142 mg, 3.36 mmol),N-diisopropylethylamine (0.71 g, 5.49 mmol) and ClPONMe₂-mo(Tr)C^(bz)(3.19 g, 4.58 mmol) were added, and the mixture was stirred at 40° C.for 16 hr. After confirmation of the completion of the reaction byUHPLC, the reaction mixture was cooled to room temperature, morpholine(0.26 g, 3.05 mmol) was added and the mixture was stirred at roomtemperature for 1 hr. The reaction mixture was ice-cooled,2,2,2-trifluoroethanol (12.4 mL) and ethanol (1.41 g, 30.5 mmol) wereadded, a solution of trifluoroacetic acid (1.74 g, 15.3 mmol) andtriethylamine (0.52 g, 5.19 mmol) in chloroform (5.8 mL) was addeddropwise, and the mixture was stirred at room temperature for 1 hr.After confirmation of the completion of the reaction by UHPLC, thereaction mixture was ice-cooled and a solution ofN,N-diisopropylethylamine (1.97 g, 15.3 mmol) in chloroform (2.7 mL) wasadded dropwise. To the reaction mixture was added acetonitrile (100 mL),and the solvent was evaporated under reduced pressure. The obtainedslurry was stirred at 0° C. for 15 min, and the precipitates werecollected by filtration. The obtained crystals were washed again withacetonitrile (50 mL), and dried under reduced pressure to giveTOB-suc-PMO[C^(bz)-T-C^(bz)] (6.74 g, yield 106%).

Example 35 Synthesis of TOB-suc-PMO[C^(bz)-T] (Lithium Chloride andAcetic Anhydride Addition System, Elongation-2)

TOB-suc-moC^(bz) (4.3 g, 3.23 mmol) was dissolved in chloroform (4.28mL), lithium chloride (150 mg, 3.55 mmol), N,N-diisopropylethylamine(0.73 g, 5.31 mmol) and ClPONMe₂-mo(Tr)T (2.95 g, 4.35 mmol) were added,and the mixture was stirred at room temperature for 15 hr. Afterconfirmation of the completion of the reaction by UHPLC, the reactionmixture was cooled to room temperature, acetic anhydride (0.03 mL, 0.32mmol) and 2,6-lutidine (0.19 mL, 1.62 mmol) were added and the mixturewas stirred at room temperature for 1 hr. To the reaction mixture wasadded morpholine (0.28 g, 3.23 mmol) and the mixture was stirred at roomtemperature for 1 hr. The reaction mixture was ice-cooled, indole (3.78g, 32.3 mmol) was added, a solution of trifluoroacetic acid (2.86 g,25.8 mmol) in chloroform (19.2 mL) was added dropwise, and the mixturewas stirred at room temperature for 1.5 hr. After confirmation of thecompletion of the reaction by UHPLC, the reaction mixture wasice-cooled, and a solution of N,N-diisopropylethylamine (3.25 g, 25.8mmol) in chloroform (4.5 mL) was added dropwise. To the reaction mixturewas added acetonitrile (86 mL) and the solvent was evaporated underreduced pressure. The obtained slurry was stirred at 0° C. for 15 min,and the precipitates were collected by filtration. The obtained crystalswere washed again with acetonitrile (43 mL) to give:TOB-Suc-PMO[C^(bz)-T]. The crystals were directly used without drying asa starting material of the next reaction.

Example 36 Synthesis of TOB-suc-PMO[C^(bz)-T-C^(bz)] (Lithium Chlorideand Acetic Anhydride Addition System, Elongation-3)

TOB-suc-PMO[C^(bz)-T] wet crystals (6.5 g, corresponding to 3.23 mmol)were dissolved in chloroform (53.4 mL), lithium chloride (150 mg, 3.36mmol), N,N-diisopropylethylamine (0.73 g, 5.81 mmol) andClPONMe₂-mo(Tr)C^(bz) (3.38 g, 4.85 mmol) were added, and the mixturewas stirred at room temperature for 40 hr. After confirmation of thecompletion of the reaction by UHPLC, the reaction mixture was cooled toroom temperature, acetic anhydride (0.03 mL, 0.32 mmol) and 2,6-lutidine(0.15 mL, 1.29 mmol) were added and the mixture was stirred at roomtemperature for 1 hr. To the reaction mixture was added morpholine (0.28g, 3.23 mmol) and the mixture was stirred at room temperature for 1 hr.The reaction mixture was ice-cooled, indole (3.78 g, 32.3 mmol) wasadded, a solution of trifluoroacetic acid (2.86 g, 25.8 mmol) inchloroform (19.2 mL) was added dropwise, and the mixture was stirred atroom temperature for 1.5 hr. After confirmation of the completion of thereaction by UHPLC, the reaction mixture was ice-cooled and a solution ofN,N-diisopropylethylamine (3.25 g, 25.8 mmol) in chloroform (4.5 mL) wasadded dropwise. To the reaction mixture was added acetonitrile (107 mL),and the solvent was evaporated under reduced pressure. The obtainedslurry was stirred at 0° C. for 15 min, and the precipitates werecollected by filtration. The obtained crystals were washed again withacetonitrile (53 mL), and dried under reduced pressure to giveTOB-suc-PMO[C^(bz)-T-C^(bz)]. The crystals were directly used withoutdrying as a starting material of the next reaction.

Example 37 Synthesis of TOB-suc-PMO[C^(bz)-C^(bz)] (Elongation-2)

TOB-suc-moC^(bz) wet crystals (16.1 g, corresponding to 3.2 mmol) weredissolved in chloroform (42 mL), N,N-diisopropylethylamine (0.74 g, 5.76mmol) and ClPONMe₂-mo(Tr)C^(bz) (3.35 g, 4.80 mmol) were added, and themixture was stirred at room temperature for 15 hr. After confirmation ofthe completion of the reaction by UHPLC, the reaction mixture was cooledto room temperature, morpholine (0.28 g, 3.2 mmol) was added and themixture was stirred at room temperature for 1 hr. The reaction mixturewas ice-cooled, 2,2,2-trifluoroethanol (14.1 mL) and ethanol (1.5 g,32.0 mmol) were added, a solution of trifluoroacetic acid (2.92 g, 25.6mmol) and triethylamine (1.13 g, 11.2 mmol) in chloroform (40 mL) wasadded dropwise, and the mixture was stirred at 15° C. for 30 min. To thereaction mixture was added 2,2,2-trifluoroethanol (3.5 mL), and themixture was further stirred at 15° C. for 1 hr. After confirmation ofthe completion of the reaction by UHPLC, the reaction mixture wasice-cooled and a solution of N,N-diisopropyl-ethylamine (3.31 g, 25.6mmol) in chloroform (30 mL) was added dropwise. To the reaction mixturewas added acetonitrile (75 mL), and the solvent was evaporated underreduced pressure. To the obtained residue was added acetonitrile (25mL), and the solvent was evaporated under reduced pressure. Acetonitrile(100 mL) was added. The obtained slurry was stirred at 0° C. for 30 min,and the precipitates were collected by filtration. The obtained crystalswere washed again with acetonitrile (100 mL), and dried under reducedpressure to give TOB-suc-PMO[C^(bz)-C^(bz)] (5.4 g, yield 96.6% relativeto TCB-suc-moC^(bz)).

Example 36 Synthesis of TOB-suc-PMO[C^(bz)-C^(bz)-T] (Elongation-3)

TOB-suc-PMO[C^(bz)-C^(bz)] (5.4 g, 3.09 mmol) was dissolved inchloroform (54 mL), N,N-diisopropylethylamine (0.72 g, 5.56 mmol) andClPONMe₂-mo(Tr)T (2.82 g, 4.64 mmol) were added, and the mixture wasstirred at room temperature for 23 hr. After confirmation of thecompletion of the reaction by UHPLC, the reaction mixture was cooled toroom temperature, morpholine (0.27 g, 3.09 mmol) was added and themixture was stirred at room temperature for 1 hr. The reaction mixturewas ice-cooled, 2,2,2-trifluoroethanol (17 mL) and ethanol (1.42 g, 30.9mmol) were added, a solution of trifluoroacetic acid (2.82 g, 24.7 mmol)and triethylamine (1.16 g, 11.4 mmol) in chloroform (39 mL) was addeddropwise, and the mixture was stirred at 15° C. for 1.5 hr. Afterconfirmation of the completion of the reaction by UHPLC, the reactionmixture was ice-cooled, and a solution of N,N-diisopropylethylamine(3.19 g, 24.7 mmol) in chloroform (30 mL) was added dropwise. To thereaction mixture was added acetonitrile (160 mL), and the solvent wasevaporated under reduced pressure. To the obtained residue was addedacetonitrile (80 mL), and the solvent was evaporated under reducedpressure. Acetonitrile (100 mL) was added. The obtained slurry wasstirred at 0° C. for 30 min, and the precipitates were collected byfiltration. The obtained crystals were washed again with acetonitrile(100 mL) and dried under reduced pressure to giveTOB-suc-PMO[C^(bz)-C^(bz)-T]. The crystals were directly used withoutdrying as a starting material of the next reaction.

Example 39 Synthesis ofTOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)-T-G^(ce/pac)-A^(bz)-A^(bz)-G^(ce/pac)-G^(ce/pac)-T-G^(ce/pac)-T-T-C^(bz)]-Tr(Results List)

TOB-suc-mo(Tr)C^(bz) (5.0 g, 3.2 mmol) was sequentially subjected tocoupling of the corresponding monomer and deprotection of trityl groupin one pot according to the methods described in Examples 37 and 38. Thestep yield at each stage is shown in the following Table. The resultantproducts with a chain length without a numerical value were used for thenext step without drying.

TABLE 8 chain length step yield  1mer 99%  2mer 97%  3mer —  4mer — 5mer 97%  6mer —  7mer —  8mer 92%  9mer — 10mer — 11mer 98% 12mer —13mer — 14mer — 15mer 96% 16mer — 17mer — 18mer 97% 19mer — 20mer 98%21mer 100% 

Example 40 Synthesis ofTOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)](Lithium Chloride Addition System, Elongation-10)

TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T] wetcrystals (12.9 g, corresponding to 2.37 mmol) were dissolved inchloroform (100 mL), lithium chloride (150 mg, 3.56 mmol),N,N-diisopropylethylamine (0.53 g, 4.27 mmol) and ClPONMe₂-mo(Tr)C^(bz)(2.48 g, 3.56 mmol) were added, and the mixture was stirred at roomtemperature for 33 hr. After confirmation of the completion of thereaction by UHPLC, the reaction mixture was cooled to room temperature,morpholine (0.78 g, 9.0 mmol) was added and the mixture was stirred atroom temperature for 1 hr. The reaction mixture was ice-cooled,2,2,2-trifluoroethanol (10.2 mL) and ethanol (1.1 g, 23.7 mmol) wereadded, and then, a solution of trifluoroacetic acid (1.85 g, 16.6 mmol)in chloroform (10 mL) was added dropwise, and the mixture was stirred atroom temperature for 2 hr.

After confirmation of the completion of the reaction by UHPLC, thereaction mixture was ice-cooled, and a solution ofN,N-diisopropylethylamine (2.07 g, 16.0 mmol) in chloroform (10 mL) wasadded dropwise. To the reaction mixture was added acetonitrile (200 mL),and the solvent was evaporated under reduced pressure. To the obtainedresidue was added acetonitrile (100 mL). The obtained slurry was stirredat 0° C. for 30 min, and the precipitates were collected by filtration.The obtained crystals were washed again with acetonitrile (100 mL) togiveTOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)].The crystals were directly used without drying as a starting material ofthe next reaction.

Example 41 Synthesis ofTOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)-T](Lithium Chloride Addition System, Elongation-11)

TOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)]wet crystals (12.6 g, 2.37 mmol) were dissolved in chloroform (110 mL),lithium chloride (150 mg, 3.56 mmol), N,N-diisopropylethylamine (0.53 g,4.27 mmol) and ClPONMe₂-mo(Tr)T (2.17 g, 3.56 mmol) were added, and themixture was stirred at room temperature for 19 hr. After confirmation ofthe completion of the reaction by UHPLC, the reaction mixture was cooledto room temperature, morpholine (0.78 g, 9.01 mmol) was added and themixture was stirred at room temperature for 1 hr. The reaction mixturewas ice-cooled, 2,2,2-trifluoroethanol (10.2 mL) and ethanol (1.09 g,23.7 mmol) were added, a solution of trifluoroacetic acid (1.89 g, 16.6mmol) in chloroform (10 mL) was added dropwise, and the mixture wasstirred at room temperature for 2 hr. After confirmation of thecompletion of the reaction by UHPLC, the reaction mixture wasice-cooled, and a solution of N,N-diisopropylethylamine (2.07 g, 16.0mmol) in chloroform (10 mL) was added dropwise. To the reaction mixturewas added acetonitrile (150 mL), and the solvent was evaporated underreduced pressure. To the obtained residue was added acetonitrile (200mL) and the solvent was evaporated under reduced pressure. Acetonitrile(50 mL) was added, and the obtained slurry was stirred at 0° C. for 30min, and the precipitates were collected by filtration. The obtainedcrystals were washed again with acetonitrile (100 mL), and dried underreduced pressure to giveTOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)-T].The crystals were directly used without drying as a starting material ofthe next reaction.

Example 42 Synthesis ofTOB-suc-PMO[C^(bz)-C^(bz)-T-C^(bz)-C^(bz)-G^(ce/pac)-G^(ce/pac)-T-T-C^(bz)-T-G^(ce/pac)-A^(bz)-A^(bz)-G^(ce/pac)-G^(ce/pac)-T-G^(ce/pac)-T-T-C^(bz)]-Tr(Results List)

TOB-suc-mo(Tr)C^(bz) (5.0 g, 3.2 mmol) was sequentially subjected tocoupling of the corresponding monomer and deprotection of trityl groupin one pot according to the methods described in Examples 37, 38, 40 and41. The step yield at each stage is shown in the following Table. Theresultant products with a chain length without a numerical value wereused for the next step without drying.

TABLE 9 chain length step yield  1mer —  2mer —  3mer —  4mer —  5mer — 6mer 89%  7mer —  8mer 91%  9mer — 10mer — 11mer — 12mer 98% 13mer —14mer 96% 15mer — 16mer — 17mer 80% 18mer — 19mer — 20mer — 21mer 69%

The scheme of Examples 43, 44 and 45 is shown below.

Example 43 Synthesis of TOB-suc-teg-Tr

3,4,5-tri(octadecyl)benzyl alcohol (91.4 mg, 0.1 mmol) was dissolved inchloroform (1.0 mL), suc-teg-Tr (72.6 mg, 0.12 mmol), EDC.HCl (23.0 mg,0.12 mmol) and 4-dimethylaminopyridine (1.2 mg, 0.01 mmol) were added,and the mixture was stirred at room temperature for 65 hr. Afterconfirmation of the completion of the reaction by UHPLC, acetonitrile (2mL) was added, and the solvent was evaporated under reduced pressure. Tothe obtained residue was added acetonitrile (4 mL), and the precipitatewas collected by filtration, and dried under reduced pressure to giveTOB-suc-teg-Tr (140 mg, yield 93%).

TOF-MS+ (m/z) 1501.0

Example 44 Synthesis of TOB-suc-teg-H

TOB-suc-teg-Tr (133 mg, 0.09 mmol) was dissolved in chloroform (1.4 mL)and ice-cooled, 2,2,2-trifluoroethanol (0.4 mL) and ethanol (42.8 g,0.93 mmol) were added, a solution of trifluoroacetic acid (82.7 mg, 0.75mmol) and triethylamine (47.4 mg, 0.47 mmol) in chloroform (0.5 mL) wasadded dropwise, and the mixture was stirred at room temperature for 2hr. After confirmation of the completion of the reaction by UHPLC, thereaction mixture was ice-cooled, and a solution ofN,N-diisopropylethylamine (97 mg, 0.75 mmol) in chloroform (0.5 mL) wasadded dropwise. To the reaction mixture was added acetonitrile (5 mL),and the solvent was evaporated: under reduced pressure. To the obtainedresidue was added acetonitrile (4 mL), and the mixture was stirred at 0°C. for 30 min. The precipitates were collected by filtration to giveTOB-suc-teg-H. The crystals were directly used without drying as astarting material of the next reaction.

TOF-MS+ (m/z) 1257.9

Example 45 Synthesis of TOB-suc-teg-mo(Tr)C^(bz)

TOB-suc-teg-H wet crystals (168 mg, corresponding to 0.09 mmol) weredissolved in chloroform (1.2 mL), N,N-diisopropylethylamine (20.7 mg,0.16 mmol) and ClPONMe₂-mo(Tr)C^(bz) (92.8 mg, 0.13 mmol) were added,and the mixture was stirred with heating at room temperature for 13 hr.After confirmation of the completion of the reaction by UHPLC, thereaction mixture was cooled to room temperature, morpholine (11.3 mg,0.09 mmol) was added and the mixture was stirred at room temperature for1 hr. To the reaction mixture was added acetonitrile (2.4 mL), and thesolvent was evaporated under reduced pressure. To the obtained residuewas added acetonitrile (3 mL), and the mixture was stirred at 0° C. for30 min. The precipitate was collected by filtration, and dried underreduced pressure to give TOB-suc-teg-mo(Tr)C^(bz) (1.48 mg, yield 86%,relative to TOB-suc-tec-Tr).

TOF-MS+ (m/z) 1919.0

INDUSTRIAL APPLICABILITY

Using a morpholino nucleotide wherein 5′-hydroxy group or a hydroxygroup present on the substituent of the 5′-hydroxy group is protected bya particular protecting group of the present invention, a method capableof efficiently producing the morpholino oligonucleotide in a high yieldby a liquid phase synthesis can be provided.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

As used heroin the words “a” and “an” and the like carry the meaning of“one or more.”

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

All patents and other references mentioned above are incorporated infull herein by this reference, the same as if set forth at length.

1-8. (canceled)
 9. A method of producing an n+p-mer morpholinooligonucleotide, comprising: (1) condensing (i) a p-mer morpholinooligonucleotide, wherein p is any integer of one or more, and wherein a5′-hydroxy group is activated (thio)phosphated or activated(thio)phosphoramidated, and a morpholine ring nitrogen atom is protectedby a temporary protecting group removable under acidic conditions, with(ii) an n-mer morpholino oligonucleotide, wherein n is an integer of oneor more, and wherein a 5′-hydroxy group or, when the 5′-hydroxy grouphas a substituent having a hydroxy group, the hydroxy group present onthe substituent is protected by a protecting group having an alkyl groupcontaining not less than 10 and not more than 300 carbon atoms and/or analkenyl group containing not less than 10 and not more than 300 carbonatoms, and the morpholine ring nitrogen atom is not protected, by a(thio)phosphoramidate bond or (thio)phosphorodiamidate bond via themorpholine ring nitrogen atom, to obtain a reaction mixture comprisingsaid n+p-mer morpholino oligonucleotide.
 10. The method according toclaim 9, wherein p is
 1. 11. The method according to claim 9, comprisingtreating said reaction mixture with a quenching agent after completionof said condensing.
 12. The method according to claim 9, furthercomprising: (1′) removing said temporary protecting group of saidmorpholine ring nitrogen atom by reacting, before said condensing (1),the n-mer morpholino oligonucleotide wherein the 5′-hydroxy group or,when the 5′-hydroxy group has a substituent having a hydroxy group, thehydroxy group present on the substituent is protected by a protectinggroup having an alkyl group containing not less than 10 and not morethan 300 carbon atoms and/or an alkenyl group containing not less than10 and not more than 300 carbon atoms, and the morpholine ring nitrogenatom is protected by a temporary protecting group removable under acidicconditions, with an acid in a non-polar solvent.
 13. The methodaccording to claim 12, wherein said temporary protecting group isremoved in the presence of a cation scavenger.
 14. The method accordingto claim 12, further comprising neutralizing with an organic base aftersaid (1′) removing said temporary protecting group of the morpholinering nitrogen atom.
 15. The method according to claim 9, wherein saidprotecting group having an alkyl group containing not less than 10 andnot more than 300 carbon atoms and or an alkenyl group containing notless than 10 and not more than 300 carbon atoms is a group representedby formula (II):Z—Y-L-   (II) wherein L is a single bond, or a group represented byformula (a1):

wherein * indicates the bonding position to Y; ** indicates the bondingposition to S¹; L₁ is an optionally substituted divalent C₁₋₂₂hydrocarbon group; and L₂ is C(═O) or a group represented by***N(R³)—R¹—N(R²)C(═O)**, wherein ** indicates the bonding position toL¹, *** indicates the bonding position to Y, R¹ is an optionallysubstituted C₁₋₂₂ alkylene group, R² and R³ are each independently ahydrogen atom or an optionally substituted C₁₋₂₂ alkyl group, or R² andR³ are optionally joined to form an optionally substituted C₁₋₂₂alkylene bond; Y is a single bond, an oxygen atom or NR, wherein R is ahydrogen atom, an alkyl group or an aralkyl group; and Z is a grouprepresented by formula (a2):

wherein * indicates the bonding position to Y; R⁴ is a hydrogen atom, orwhen R_(b) is a group represented by the following formula (a3), R⁴ isoptionally a single bond or —O— in combination with R⁶ to form afluorenyl group or a xanthenyl group together with ring B; each of k Qis independently a single bond, or —O—, —S—, —OC(═O)—, —NHC(═O)— or—NH—; each of k R⁵ is independently an organic group having an alkylgroup containing not less than 10 and not more than 300 carbon atomsand/or an alkenyl group containing not less than 10 and not more than300 carbon atoms; k is an integer of 1 to 4; ring A optionally furtherhas, in addition to k of QR⁵, a substituent selected from the groupconsisting of a halogen atom, a C₁₋₆ alkyl group optionally substitutedby one or more halogen atoms, and a C₁₋₆ alkoxy group optionallysubstituted by one or more halogen atoms; R_(a) is a hydrogen atom;R_(b) is a hydrogen atom, or a group represented by formula (a3):

wherein * indicates a bonding position; j is an integer of 0 to 4; eachof j Q is as defined above; each of j R⁷ is independently an organicgroup having an alkyl group containing not less than 10 and not morethan 300 carbon atoms and/or an alkenyl group containing not less than10 and not more than 300 carbon atoms; R⁶ is a hydrogen atom, oroptionally a single bond or —O— in combination with R⁴ to form afluorenyl group or a xanthenyl group together with ring A; and ring Boptionally further has, in addition to jo QR⁷, a substituent selectedfrom the group consisting of a halogen atom, a C₁₋₆ alkyl groupoptionally substituted by one or more halogen atoms, and a C₁₋₆ alkoxygroup optionally substituted by one or more halogen atoms, or R_(a) andR_(b) are joined to form an oxygen atom.
 16. The method according toclaim 15, further comprising: (2) obtaining said morpholinooligonucleotide by adding a polar solvent to said reaction mixtureobtained in step (1) and/or (1′) and collecting a precipitate thereof bysolid-liquid separation.
 17. The method according to claim 12, whereinsaid non-polar solvent is selected from the group consisting of ahalogenated solvent, an aromatic solvent, an ester solvent, an aliphaticsolvent, a non-polar ether solvent, and a combination thereof.
 18. Themethod according to claim 16, wherein said polar solvent is an alcoholsolvent or a nitrile solvent.
 19. The method according to claim 9,further comprising: (3) removing all protecting groups of said n+p-mermorpholino oligonucleotide.
 20. The method according to claim 9, whereinsaid temporary protecting group removable under acidic conditions is atrityl group, a dimethoxytrityl group, or a monomethoxytrityl group.